Use of oxyhydrogen microorganisms for non-photosynthetic carbon capture and conversion of inorganic and/or c1 carbon sources into useful organic compounds

ABSTRACT

Compositions and methods for a hybrid biological and chemical process that captures and converts carbon dioxide and/or other forms of inorganic carbon and/or CI carbon sources including but not limited to carbon monoxide, methane, methanol, formate, or formic acid, and/or mixtures containing CI chemicals including but not limited to various syngas compositions, into organic chemicals including bio-fuels or other valuable biomass, chemical, industrial, or pharmaceutical products are provided. The present invention, in certain embodiments, fixes inorganic carbon or CI carbon sources into longer carbon chain organic chemicals by utilizing microorganisms capable of performing the oxyhydrogen reaction and the autotrophic fixation of CO 2  in one or more steps of the process.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/328,184, filed Apr. 27, 2010 andentitled “USE OF OXYHYDROGEN MICROORGANISMS FOR NON-PHOTOSYNTHETICCARBON CAPTURE AND CONVERSION OF INORGANIC CARBON SOURCES INTO USEFULORGANIC COMPOUNDS.” This application is also a continuation-in-part ofInternational Patent Application No. PCT/US2010/001402, filed May 12,2010, and entitled “BIOLOGICAL AND CHEMICAL PROCESS UTILIZINGCHEMOAUTOTROPHIC MICROORGANISMS FOR THE CHEMOSYNTHETIC FIXATION OFCARBON DIOXIDE AND/OR OTHER INORGANIC CARBON SOURCES INTO ORGANICCOMPOUNDS, AND THE GENERATION OF ADDITIONAL USEFUL PRODUCTS,” which is acontinuation-in-part of U.S. patent application Ser. No. 12/613,550,filed Nov. 6, 2009, and entitled “BIOLOGICAL AND CHEMICAL PROCESSUTILIZING CHEMOAUTOTROPHIC MICROORGANISMS FOR THE CHEMOSYNTHETICFIXATION OF CARBON DIOXIDE AND/OR OTHER INORGANIC CARBON SOURCES INTOORGANIC COMPOUNDS, AND THE GENERATION OF ADDITIONAL USEFUL PRODUCTS,”which claims the benefit of U.S. Provisional Patent Application No.61/111,794, filed Nov. 6, 2008, and entitled, “BIOLOGICAL AND CHEMICALPROCESS UTILIZING CHEMOAUTOTROPHIC MICROORGANISMS FOR THE RECYCLING OFCARBON FROM CARBON DIOXIDE AND OTHER INORGANIC CARBON SOURCES THROUGHCHEMOSYNTHESIS INTO BIOFUEL AND ADDITIONAL USEFUL PRODUCTS.” Each ofthese applications is incorporated herein by reference in its entiretyfor all purposes.

FIELD OF INVENTION

The present invention falls within the technical areas of biofuels,bioremediation, carbon capture, carbon dioxide-to-fuels, carbonrecycling, carbon sequestration, energy storage, gas-to-liquids, wasteenergy to fuels, syngas conversions, and renewable/alternative and/orlow carbon dioxide emission sources of energy. Specifically the presentinvention is a unique example of the use of biocatalysts within abiological and chemical process to fix carbon dioxide and/or other formsof inorganic carbon and/or or other C1 carbon sources into longer carbonchain organic chemical products in a non-photosynthetic process poweredby low carbon emission energy sources and/or waste energy sources. Inaddition the present invention involves the production of chemicalco-products that are co-generated through carbon-fixation reaction stepsand/or non-biological reaction steps as part of an overall carboncapture and conversion process or syngas conversion process. The presentinvention can enable the effective and economic capture of carbondioxide from the atmosphere or from a point source of carbon dioxideemissions as well as the economic use of waste energy sources and/orrenewable energy sources and/or low carbon emission energy sources, forthe production of liquid transportation fuel and/or other organicchemical products, which will help address greenhouse gas inducedclimate change and contribute to the domestic production of renewableliquid transportation fuels and/or other organic chemicals without anydependence upon agriculture.

BACKGROUND

Great interest and resources have been directed towards developingtechnologies that use renewable energy or waste energy for theconversion of carbon dioxide, or other low value carbon sources, intouseful organic chemicals in order to provide alternatives to chemicals,materials and fuels derived from petroleum or other fossil sources. Mostof the focus in the area of CO₂ conversion has been placed on biologicalapproaches that utilize photosynthesis to fix CO₂ into biomass orend-products, while some effort has been directed at fully abiotic andchemical processes for fixing CO₂.

A type of CO₂-to-organic chemical approach that has received relativelyless attention is hybrid chemical/biological processes where thebiological step is limited to CO₂ fixation alone, corresponding to thedark reaction of photosynthesis. The potential advantages of such ahybrid CO₂-to-organic chemical process include the ability to combineenzymatic capabilities gained through billions of years of evolution infixing CO₂, with a wide array of abiotic technologies to power theprocess such as solar PV, solar thermal, wind, geothermal,hydroelectric, or nuclear. Microorganisms performing carbon fixationwithout light can be contained in more controlled and protectedenvironments, less prone to water and nutrient loss, contamination, orweather damage, than what can be used for culturing photosyntheticmicroorganisms. Furthermore an increase in bioreactor capacity can bemet with vertical rather than horizontal construction, making itpotentially far more land efficient. A hybrid chemical/biological systemoffers the possibility of a CO₂-to-organic chemical process that avoidsmany drawbacks of photosynthesis while retaining the biologicalcapabilities for complex organic synthesis from CO₂.

Chemoautotrophic microorganisms are generally microbes that can performCO₂ fixation like in the photosynthetic dark reaction, but which can getthe reducing equivalents needed for CO₂ fixation from an inorganicexternal source, rather than having to internally generate them throughthe photosynthetic light reaction. Carbon fixing biochemical pathwaysthat occur in chemoautotrophs include the reductive tricarboxylic acidcycle, the Calvin-Benson-Bassham cycle, and the Wood-Ljungdahl pathway.

Prior work is known relating to certain applications of chemoautotrophicmicroorganisms in the capture and conversion of CO₂ gas to fixed carbon.However, many of these approaches have suffered shortcomings that havelimited the effectiveness, economic feasibility, practicality andcommercial adoption of the described processes. The present invention incertain aspects addresses one or more of the aforementioned shortcomings

It is believed that the present invention utilizing oxyhydrogenmicroorganisms in the chemosynthetic fixation of CO₂ under carefullycontrolled oxygen levels may have advantages for the production oflonger chain organic compounds (e.g., C₅ and longer). The ability toproduce longer chain organic compounds is an important advantage for thepresent invention since the energy densities (energy per unit volume)are generally higher for longer chain organic compounds, and thecompatibility with the current transportation fleet is generally greaterrelative to, for example, shorter chain products such as C1 and C2products.

SUMMARY OF THE INVENTION

In response to a need in the art that the inventors have recognized inmaking the invention, a novel combined biological and chemical processfor the capture and conversion of inorganic carbon and/or C1 carbonsources to longer chain organic compounds, and particularly organiccompounds with C5 or longer chain lengths, through the use ofoxyhydrogen microorganisms for carbon capture and fixation is described.In some embodiments, the process can couple the efficient production ofhigh value organic compounds such as liquid hydrocarbon fuel with thedisposal of waste sources of carbon, as well as with the capture of CO₂,which can generate additional revenue.

In one aspect, a biological and chemical method for the capture andconversion of an inorganic carbon compound and/or an organic compoundcontaining only one carbon atom into an organic chemical product isdescribed. In some embodiments, the method comprises introducing aninorganic carbon compound and/or an organic compound containing only onecarbon atom into an environment suitable for maintaining oxyhydrogenmicroorganisms and/or capable of maintaining extracts of oxyhydrogenmicroorganisms; and converting the inorganic carbon compound and/or theorganic compound containing only one carbon atom into the organicchemical product and/or a precursor thereof within the environment viaat least one chemosynthetic carbon-fixing reaction utilizing theoxyhydrogen microorganisms and/or cell extracts containing enzymes fromthe oxyhydrogen microorganisms. In some embodiments, the chemosyntheticfixing reaction is at least partially driven by chemical and/orelectrochemical energy provided by electron donors and electronacceptors that have been generated chemically and/or electrochemicallyand/or are introduced into the environment from at least one sourceexternal to the environment.

In one aspect, a bioreactor is described. The bioreactor comprises, inone set of embodiments, a first column comprising an upper portion and alower portion; and a second column comprising an upper portion and alower portion, the upper portion of the second column fluidicallyconnected to the upper portion of the first column, and the lowerportion of the second column fluidically connected to the lower portionof the first column In some embodiments, the bioreactor is constructedand arranged such that, when a liquid is circulated between the firstand second columns, a volume of gas is substantially stationary at thetop of the first column and/or the second column In some embodiments,the volume of gas occupies at least about 2% of the total volume of thecolumn in which the volume is positioned.

In another aspect, a method of operating a bioreactor is provided. Themethod comprises, in some embodiments, circulating a liquid comprising agrowth medium between a first column and a second column, wherein,during operation, a volume of gas remains substantially stationary atthe top of the first column and/or the second column, and the volume ofgas occupies at least about 2% of the total volume of the column inwhich the volume is positioned.

In one aspect, an electrolysis device is provided. In some embodiments,the electrolysis device comprises a chamber constructed and arranged toelectrolyze water to produce oxygen and hydrogen; and an outletcomprising a separator constructed and arranged to separate at least aportion of the oxygen within a stream from at least a portion of thehydrogen within a stream such that the hydrogen content of the fluidexiting the separator is suitable for use as a feed stream to a reactorcontaining a culture of oxyhydrogen microorganisms.

In another aspect, a method of operating an electrolysis device isdescribed. The method comprises, in some embodiments, electrolyzingwater to produce a first stream containing oxygen and hydrogen; andseparating at least a portion of the oxygen from at least a portion ofthe hydrogen to produce a second stream relatively rich in hydrogencompared to the first stream, wherein the second stream is suitable foruse as a feed stream to a reactor containing a culture of oxyhydrogenmicroorganisms.

The present invention, in certain embodiments, provides compositions andmethods for the capture of carbon dioxide from carbon dioxide-containinggas streams and/or atmospheric carbon dioxide or carbon dioxide indissolved, liquefied or chemically-bound form through a chemical andbiological process that utilizes obligate or facultative oxyhydrogenmicroorganisms, and/or cell extracts containing enzymes from oxyhydrogenmicroorganisms in one or more carbon fixing process steps.

The present invention, in certain embodiments, provides compositions andmethods for the utilization of C1 carbon sources including but notlimited to carbon monoxide, methane, methanol, formate, or formic acid,and/or mixtures containing C1 chemicals including but not limited tovarious syngas compositions generated from various gasified, pyrolyzed,or steam-reformed fixed carbon feedstocks, and convert said C1 chemicalsinto longer chain organic compounds,

The present invention, in certain embodiments, provides compositions andmethods for the recovery, processing, and use of the organic compoundsproduced by chemosynthetic reactions performed by oxyhydrogenmicroorganisms to fix inorganic carbon and/or C1 carbon sources intolonger chain organic compounds. The present invention, in certainembodiments, provides compositions and methods for the maintenance andcontrol of the oxygen levels in the carbon-fixation environment for theenhanced (e.g., optimal) production of C5 or longer organic compoundproducts through carbon fixation. The present invention, in certainembodiments, provides compositions and methods for the generation,processing and delivery of chemical nutrients needed for carbon-fixationand maintenance of oxyhydrogen microorganism cultures, including but notlimited to the provision of electron donors and electron acceptorsneeded for non-photosynthetic carbon-fixation. The present invention, incertain embodiments, provides compositions and methods for themaintenance of an environment conducive for carbon-fixation, and therecovery and recycling of unused chemical nutrients and process water.

The present invention, in certain embodiments, provides compositions andmethods for chemical process steps that occur in series and/or inparallel with the chemosynthetic reaction steps that: convert unrefinedraw input chemicals to more refined chemicals that are suited forsupporting the chemosynthetic carbon fixing step; that convert energyinputs into a chemical form that can be used to drive chemosynthesis,and specifically into chemical energy in the form of electron donors andelectron acceptors; that direct inorganic carbon captured fromindustrial or atmospheric or aquatic sources to the carbon fixationsteps of the process under conditions that are suitable to supportchemosynthetic carbon fixation by the oxyhydrogen microorganisms orenzymes and/or direct C1 chemicals derived from low value or wastesources of carbon such as carbon monoxide, methane, methanol, formate,or formic acid, and/or mixtures containing C1 chemicals including butnot limited to various syngas compositions derived from thegasification, pyrolysis, or steam reforming of various low value orwaste carbon sources, that can be used by the oxyhydrogen microorganismas a carbon sources and any energy source for the synthesis of longerchain organic chemicals; that further process the output products of thecarbon fixation steps into a form suitable for storage, shipping, andsale, and/or safe disposal in a manner that results in a net reductionof gaseous CO₂ released into the atmosphere and/or the upgrade of a lowvalue or waste material into a finished chemical, fuel, or nutritionalproduct. The fully chemical process steps combined with thechemosynthetic carbon fixation steps constitute the overall carboncapture and conversion process of some embodiments of the presentinvention.

One feature of certain embodiments of the present invention is theinclusion of one or more process steps within a chemical process for thecapture of inorganic carbon and conversion to fixed carbon products,that utilize oxyhydrogen microorganisms and/or enzymes from oxyhydrogenmicroorganisms as a biocatalyst for the fixation of carbon dioxide incarbon dioxide-containing gas streams or the atmosphere or water and/ordissolved or solid forms of inorganic carbon, into organic compounds. Insome such embodiments carbon dioxide containing flue gas, or processgas, or air, or inorganic carbon in solution as dissolved carbondioxide, carbonate ion, or bicarbonate ion including aqueous solutionssuch as sea water, or inorganic carbon in solid phases such as but notlimited to carbonates and bicarbonates, is pumped or otherwise added toa vessel or enclosure containing nutrient media and oxyhydrogenmicroorganisms. In some such cases oxyhydrogen microorganisms performchemosynthesis to fix inorganic carbon into organic compounds using thechemical energy stored in molecular hydrogen and/or valence orconduction electrons in solid state electrode materials and/or one ormore of the following list of electron donors pumped or otherwiseprovided to the nutrient media including but not limited to: ammonia;ammonium; carbon monoxide; dithionite; elemental sulfur; hydrocarbons;metabisulfites; nitric oxide; nitrites; sulfates such as thiosulfatesincluding but not limited to sodium thiosulfate (Na₂S₂O₃) or calciumthiosulfate (CaS₂O₃); sulfides such as hydrogen sulfide; sulfites;thionate; thionite; transition metals or their sulfides, oxides,chalcogenides, halides, hydroxides, oxyhydroxides, phosphates, sulfates,or carbonates, in soluble or solid phases. In some embodiments,conduction or valence band electrons in solid state electrode materialscan be used. The electron donors can be oxidized by electron acceptorsin the chemosynthetic reaction. Electron acceptors that may be used atthe chemosynthetic reaction step include oxygen and/or other electronacceptors including but not limited to one or more of the following:carbon dioxide, ferric iron or other transition metal ions, nitrates,nitrites, sulfates, oxygen, or valence or conduction band holes in solidstate electrode materials.

One feature of certain embodiments of the present invention is theinclusion of one or more process steps within a chemical process for theconversion of C1 carbon sources including but not limited to carbonmonoxide, methane, methanol, formate, or formic acid, and/or mixturescontaining C1 chemicals including but not limited to various syngascompositions generated from various gasified, pyrolyzed, orsteam-reformed fixed carbon feedstocks, that utilize oxyhydrogenmicroorganisms and/or enzymes from oxyhydrogen microorganisms as abiocatalyst for the conversion of C1 chemicals into longer chain organicchemicals (i.e. C2 or longer and, in some embodiments, C5 or longercarbon chain molecules). In some such embodiments C1 containing syngas,or process gas, or C1 chemicals in a pure liquid form or dissolved insolution is pumped or otherwise added to a vessel or enclosurecontaining nutrient media and oxyhydrogen microorganisms. In some suchcases oxyhydrogen microorganisms perform biochemical synthesis toelongate C1 chemicals into longer carbon chain organic chemicals usingthe chemical energy stored in the C1 chemical, and/or molecular hydrogenand/or valence or conduction electrons in solid state electrodematerials and/or one or more of the following list of electron donorspumped or otherwise provided to the nutrient media including but notlimited to: ammonia; ammonium; carbon monoxide; dithionite; elementalsulfur; hydrocarbons; metabisulfites; nitric oxide; nitrites; sulfatessuch as thiosulfates including but not limited to sodium thiosulfate(Na₂S₂O₃) or calcium thiosulfate (CaS₂O₃); sulfides such as hydrogensulfide; sulfites; thionate; thionite; transition metals or theirsulfides, oxides, chalcogenides, halides, hydroxides, oxyhydroxides,sulfates, or carbonates, in soluble or solid phases. The electron donorscan be oxidized by electron acceptors in a chemosynthetic reaction.Electron acceptors that may be used at this reaction step include oxygenand/or other electron acceptors including but not limited to one or moreof the following: carbon dioxide, ferric iron or other transition metalions, nitrates, nitrites, oxygen, or holes in solid state electrodematerials.

The chemosynthetic reaction step or steps of the process whereby carbondioxide and/or inorganic carbon is fixed into organic carbon in the formof organic compounds and biomass and/or the reaction steps converting C1chemicals to longer chain organic chemicals whereby a C1 chemical suchas but not limited to carbon monoxide, methane, methanol, formate, orformic acid, and/or mixtures containing C1 chemicals including but notlimited to various syngas compositions generated from various gasified,pyrolyzed, or steam-reformed fixed carbon feedstocks, are biochemicallyconverted into longer chain organic chemicals (i.e. C2 or longer and, insome embodiments, C5 or longer carbon chain molecules) can be performedin aerobic, microaerobic, anoxic, anaerobic conditions, or facultativeconditions. A facultative environment is considered to be one havingaerobic upper layers and anaerobic lower layers caused by stratificationof the water column.

The oxygen level is controlled in some embodiments of the currentinvention so that the production of targeted organic compounds by theoxyhydrogen microorganisms through carbon-fixation is controlled (e.g.,optimized). One objective of controlling oxygen levels is to control(e.g., optimize) the intracellular Adenosine Triphosphate (ATP)concentration through the cellular reduction of oxygen and production ofATP by oxidative phosphorylation, while simultaneously keeping theenvironment sufficiently reducing so that a high ratio of NADH (orNADPH) to NAD (or NADP) is also maintained.

An advantage of using oxyhydrogen microorganisms over strictly anaerobicacetogenic or methanogenic microorganisms for carbon captureapplications and/or syngas conversion applications is the higher oxygentolerance of oxyhydrogen microorganisms.

A further advantage of using oxyhydrogen microorganisms for carboncapture applications and/or syngas conversion applications and/orbiofuel production over using acetogens is that the production of ATPpowered by the oxyhydrogen reaction results in a water product, whichcan readily be incorporated into the process stream, rather than thegenerally undesirable acetic acid or butyric acid products ofacidogenesis which can harm the microorganisms by dropping the solutionpH or accumulating to toxic levels.

An additional feature of certain embodiments of the present inventionregards the source, production, or recycling of the electron donors usedby the oxyhydrogen microorganisms to fix carbon dioxide into organiccompounds and/or to synthesize longer carbon chain organic moleculesfrom C1 chemicals. The electron donors used for carbon dioxide captureand carbon fixation can be produced or recycled in certain embodimentsof the present invention electrochemically or thermochemically usingpower from a number of different renewable and/or low carbon emissionenergy technologies including but not limited to: photovoltaics, solarthermal, wind power, hydroelectric, nuclear, geothermal, enhancedgeothermal, ocean thermal, ocean wave power, tidal power. The electrondonors can also be of mineralogical origin including but not limited toreduced S and Fe containing minerals. The electron donors used incertain embodiments of the present invention can also be produced orrecycled through chemical reactions with hydrocarbons that may or maynot be a non-renewable fossil fuel, but where said chemical reactionsproduce low or zero carbon dioxide gas emissions. For example oxidereduction reactions that produce a carbonate and a hydrogen product thatcan be used as electron donor in the carbon-fixation reaction steps ofcertain embodiments of the present invention include:

2CH₄+Fe₂O₃+3H₂O→2FeCO₃+7H₂

and/or

CH₄+CaO+2H₂O→CaCO₃+4H₂.

An additional feature of certain embodiments of the present inventionregards the formation and recovery of organic compounds and/or biomassfrom the chemosynthetic carbon fixation step or steps. These organiccompounds and/or biomass products can have a variety of applications.

An additional feature of certain embodiments of the present inventionregards using modified oxyhydrogen microorganisms in the carbon-fixationstep/steps such that a superior quantity and/or quality of organiccompounds, biochemicals, or biomass is generated through chemosynthesis.The oxyhydrogen microbes used in these steps may be modified throughartificial means including but not limited to accelerated mutagenesis(e.g. using ultraviolet light or chemical treatments), geneticengineering or modification, hybridization, synthetic biology ortraditional selective breeding. Possible modifications of theoxyhydrogen microorganisms include but are not limited to those directedat producing increased quantity and/or quality of organic compoundsand/or biomass to be used as a biofuels, or as feedstock for theproduction of biofuels including, but not limited to JP-8 jet fuel,diesel, gasoline, biodiesel, butanol, ethanol, hydrocarbons, methane,and pseudovegetable oil or any other hydrocarbon suitable for use as arenewable/alternate fuel leading to lowered greenhouse gas emissions.

Also described are compositions and methods that reduce the hazards ofperforming gas fermentations that utilize mixtures of hydrogen andoxygen within the invented process.

Compositions and methods that take advantage of the oxygen tolerance andability to use oxygen as an electron acceptor possessed by oxyhydrogenmicroorganisms in order to enable a system for converting water intohydrogen or hydride electron donors and oxygen electron acceptors, thathas improved efficiency over the application of current state-of-the-artelectrolysis for the purpose of generating hydrogen or hydride electrondonors and oxygen electron acceptors, are also described.

Also described are process steps for the recovery and further finishingof useful chemicals produced both by the biological carbon fixationsteps of the process, as well as from non-biological process steps.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. All publications, patent applications and patentsmentioned in the text are incorporated by reference in their entirety.In cases where the present specification and a document incorporated byreference include conflicting and/or inconsistent disclosure, thepresent specification shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. For purposes ofclarity, not every component is labeled in every figure, nor is everycomponent of each embodiment of the invention shown where illustrationis not necessary to allow those of ordinary skill in the art tounderstand the invention. In the figures:

FIG. 1 is a general process flow diagram for one embodiment of thisinvention for a carbon capture and fixation process;

FIG. 2 is a process flow diagram for another embodiment of the presentinvention with capture of CO₂ performed by a microorganism capable ofperforming an oxyhydrogen reaction (e.g., hydrogen oxidizing purplenon-sulfur bacteria) to produce a lipid-rich biomass that is convertedinto JP-8 jet fuel;

FIG. 3 is diagram of a bioreactor design that can avoid dangerousmixtures of hydrogen and oxygen by exploiting the low solubilities ofhydrogen and oxygen gas in water while providing the oxyhydrogenmicroorganism with the oxygen and hydrogen needed for cellular energyand carbon fixation;

FIG. 4 is a diagram of a bioreactor design that takes advantage of therelatively high solubility of carbon dioxide and the strong ability ofoxyhydrogen microorganism to capture carbon dioxide from relativelydilute streams using a carbon concentrating mechanism (CCM), to removeCO₂ from a dilute gas mixture and separate it from low solubility gasessuch as oxygen and nitrogen; and

FIG. 5 is an electrolysis technology that is specially designed to takeadvantage of the oxyhydrogen microorganisms' tolerance and need for acertain concentration of oxygen by decreasing the complete separation ofthe hydrogen and oxygen produced from standard electrolysis.

DETAILED DESCRIPTION

The present invention provides, in certain embodiments, compositions andmethods for the capture and fixation of carbon dioxide from carbondioxide-containing gas streams and/or atmospheric carbon dioxide orcarbon dioxide in liquefied or chemically-bound form through a chemicaland biological process that utilizes obligate or facultative oxyhydrogenmicroorganisms, and/or cell extracts containing enzymes from oxyhydrogenmicroorganisms in one or more process steps. The fixation of inorganiccarbon sources other than CO₂ and/or other C1 carbon sources are alsodescribed. Cell extracts include but are not limited to: a lysate,extract, fraction or purified product exhibiting chemosynthetic enzymeactivity that can be created by standard methods from oxyhydrogenmicroorganisms. In addition the present invention, in certainembodiments, provides compositions and methods for the recovery,processing, and use of the chemical products of chemosynthetic reactionstep or steps performed by oxyhydrogen microorganisms to fix inorganiccarbon into organic compounds and/or synthetic reaction step or stepsperformed by oxyhydrogen microorganisms to elongate C1 molecules tolonger carbon chain organic chemicals. Finally the present invention, incertain embodiments, provides compositions and methods for theproduction and processing and delivery of chemical nutrients needed forchemoautotrophic carbon-fixation by the oxyhydrogen microorganisms, andparticularly electron donors including but not limited to molecularhydrogen and/or electrical power, and electron acceptors including butnot limited to oxygen and carbon dioxide to drive the carbon fixationreaction; compositions and methods for the maintenance of an environmentconducive for carbon-fixation by oxyhydrogen microorganisms; andcompositions and methods for the removal of the chemical products ofchemosynthesis from the oxyhydrogen culture environment and the recoveryand recycling of unused of chemical nutrients.

The terms “molecular hydrogen,” “dihydrogen,” and “H₂” are usedinterchangeably throughout.

The terms “oxyhydrogen microorganism” and “knallgas microorganism” areused interchangeably throughout. Oxyhydrogen microorganisms aregenerally described in Chapter 5, Section III of Thermophilic Bacteria,a book by Jakob Kristjansson, CRC Press, 1992, which is incorporatedherein by reference. Generally, oxyhydrogen microorganisms are capableof performing the oxyhydrogen reaction. Oxyhydrogen microorganismsgenerally have the ability to use molecular hydrogen by means ofhydrogenases with some of the electrons donated from H₂ being utilizedfor the reduction of NAD⁺ (and/or other intracellular reducingequivalents) and the rest of the electrons for aerobic respiration. Inaddition, oxyhydrogen microorganisms generally are capable of fixing CO₂autotrophically, through pathways such as the reverse Calvin Cycle orthe reverse citric acid cycle.

In addition, the terms “oxyhydrogen reaction” and “knallgas reaction”are used interchangeably throughout to refer to the microbial oxidationof molecular hydrogen by molecular oxygen. The oxyhydrogen reaction isgenerally expressed as:

2H₂+O₂→2H₂O+energy

and/or by stoichiometric equivalents of this reaction.

Exemplary oxyhydrogen microorganisms that can be used in one or moreprocess steps of certain embodiments of the present invention includebut are not limited to one or more of the following: purple non-sulfurphotosynthetic bacteria including but not limited to Rhodopseudomonaspalustris, Rhodopseudomonas capsulata, Rhodopseudomonas viridis,Rhodopseudomonas sulfoviridis, Rhodopseudomonas blastica,Rhodopseudomonas spheroides, Rhodopseudomonas acidophila and otherRhodopseudomonas sp., Rhodospirillum rubrum, and other Rhodospirillumsp.; Rhodococcus opacus and other Rhodococcus sp.; Rhizobium japonicumand other Rhizobium sp.; Thiocapsa roseopersicina and other Thiocapsasp.; Pseudomonas hydrogenovora, Pseudomonas hydrogenothermophila, andother Pseudomonas sp.; Hydrogenomonas pantotropha, Hydrogenomonaseutropha, Hydrogenomonas facilis, and other Hydrogenomonas sp.;Hydrogenobacter thermophilus and other Hydrogenobacter sp.;Hydrogenovibrio marinus and other Hydrogenovibrio sp.; Helicobacterpylori and other Helicobacter sp.; Xanthobacter sp.; Hydrogenophaga sp.;Bradyrhizobium japonicum and other Bradyrhizobium sp.; Ralstoniaeutropha and other Ralstonia sp.; Alcaligenes eutrophus and otherAlcaligenes sp.; Variovorax paradoxus, and other Variovorax sp.;Acidovorax facilis, and other Acidovorax sp.; cyanobacteria includingbut not limited to Anabaena oscillarioides, Anabaena spiroides, Anabaenacylindrica, and other Anabaena sp.; green algae including but notlimited to Scenedesmus obliquus and other Scenedesmus sp., Chlamydomonasreinhardii and other Chlamydomonas sp., Ankistrodesmus sp., Rhaphidiumpolymorphium and other Rhaphidium sp.; as well as a consortiums ofmicroorganisms that include oxyhydrogen microorganisms.

The different oxyhydrogen microorganisms that can be used in certainembodiments of the present invention may be native to a rangeenvironments including but not limited to hydrothermal vents, geothermalvents, hot springs, cold seeps, underground aquifers, salt lakes, salineformations, mines, acid mine drainage, mine tailings, oil wells,refinery wastewater, oil, gas, or hydrocarbon contaminated waters; coalseams, the deep sub-surface, waste water and sewage treatment plants,geothermal power plants, sulfatara fields, soils including but notlimited to soils contaminated with hydrocarbons and/or located under oraround oil or gas wells, oil refineries, oil pipelines, gasoline servicestations. They may or may not be extremophiles including but not limitedto thermophiles, hyperthermophiles, acidophiles, halophiles, andpsychrophiles.

In some embodiments, relatively long-chain chemical products can beproduced. For example, the organic chemical product produced in someembodiments can include compounds with carbon chain lengths of at leastC5, at least C10, at least C15, at least C20, between about C5 and aboutC30, between about C10 and about C30, between about C15 and about C30,or between about C20 and about C30.

FIG. 1 illustrates the general process flow diagram for embodiments ofthe present invention that have a process step for the generation ofelectron donors (e.g., molecular hydrogen electron donors) suitable forsupporting chemosynthesis from an energy input and raw inorganicchemical input; followed by recovery of chemical co-products from theelectron donor generation step; delivery of generated electron donorsalong with oxygen electron acceptors, water, nutrients, and CO₂ from apoint industrial flue gas source, into chemosynthetic reaction step orsteps that make use of oxyhydrogen microorganisms to capture and fixcarbon dioxide, creating chemical and biomass co-products throughchemosynthetic reactions; followed by process steps for the recovery ofboth chemical and biomass products from the process stream; andrecycling of unused nutrients and process water, as well as cell massneeded to maintain the microbial culture, back into the carbon-fixationreaction steps.

In the embodiment illustrated in FIG. 1, the CO₂ containing flue gas iscaptured from a point source or emitter. Electron donors (e.g., H₂)needed for chemosynthesis can be generated from input inorganicchemicals and energy. The flue gas can be pumped through bioreactorscontaining oxyhydrogen microorganisms along with electron donors andacceptors needed to drive chemosynthesis and a medium suitable tosupport the microbial culture and carbon fixation throughchemosynthesis. The cell culture may be continuously flowed into and outof the bioreactors. After the cell culture leaves the bioreactors, thecell mass can be separated from the liquid medium. Cell mass needed toreplenish the cell culture population at a desirable (e.g., optimal)level can be recycled back into the bioreactor. Surplus cell mass can bedried to form a dry biomass product which can be further post-processedinto various chemical, fuel, or nutritional products. Following the cellseparation step, extracellular chemical products of the chemosyntheticreaction can be removed from the process flow and recovered. Then, anyundesirable waste products that might be present are removed. Followingthis, the liquid medium and any unused nutrients can be recycled backinto the bioreactors.

Many of the reduced inorganic chemicals upon which chemoautotrophs grow(e.g. H₂, H₂S, ferrous iron, ammonium, Mn²⁺) can be readily producedusing electrochemical and/or thermochemical processes known in the artof chemical engineering that may optionally be powered by a varietycarbon dioxide emission-free or low-carbon emission and/or renewablesources of power including wind, hydroelectric, nuclear, photovoltaics,or solar thermal.

Certain embodiments of the present invention use carbon dioxideemission-free or low-carbon emission and/or renewable sources of powerin the production of electron donors including but not limited to one ormore of the following: photovoltaics, solar thermal, wind power,hydroelectric, nuclear, geothermal, enhanced geothermal, ocean thermal,ocean wave power, tidal power. In certain embodiments of the presentinvention oxyhydrogen microorganisms function as biocatalysts for theconversion of renewable energy and/or low or zero carbon emission energyinto liquid hydrocarbon fuel, or high energy density organic compoundsgenerally, with CO₂ captured from flue gases, or from the atmosphere, orocean serving as a carbon source. These embodiments of the presentinvention can provide renewable energy technologies with the capabilityof producing a transportation fuel having significantly higher energydensity than if the renewable energy sources are used to producehydrogen gas—which must be stored in relatively heavy storage systems(e.g. tanks or storage materials)—or if it is used to charge batteries,which have relatively low energy density. Additionally the liquidhydrocarbon fuel product of certain embodiments of the present inventionmay be more compatible with the current transportation infrastructurecompared to battery or hydrogen energy storage options.

The position of the process step or steps for the generation of electrondonors (e.g., molecular hydrogen electron donors) in the general processflow of certain embodiments of the present invention is illustrated inFIG. 1 by Box 3, labeled “Electron Donor Generation.” Electron donorsproduced in certain embodiments of the present invention usingelectrochemical and/or thermochemical processes known in the art ofchemical engineering and/or generated from natural sources include, butare not limited to molecular hydrogen and/or valence or conductionelectrons in solid state electrode materials and/or other reducingagents including but are not limited to one or more of the following:ammonia; ammonium; carbon monoxide; dithionite; elemental sulfur;hydrocarbons; metabisulfites; nitric oxide; nitrites; sulfates such asthiosulfates including but not limited to sodium thiosulfate (Na₂S₂O₃)or calcium thiosulfate (CaS₂O₃); sulfides such as hydrogen sulfide;sulfites; thionate; thionite; transition metals or their sulfides,oxides, chalcogenides, halides, hydroxides, oxyhydroxides, sulfates, orcarbonates, in soluble or solid phases.

Certain embodiments of the present invention use molecular hydrogen asthe electron donor. Hydrogen electron donors are generated by methodsknown in to art of chemical and process engineering including but notlimited to one or more of the following: through electrolysis of waterby approaches including but not limited to using Proton ExchangeMembranes (PEM), liquid electrolytes such as KOH, high-pressureelectrolysis, high temperature electrolysis of steam (HTES);thermochemical splitting of water through methods including but notlimited to the iron oxide cycle, cerium(IV) oxide-cerium(III) oxidecycle, zinc zinc-oxide cycle, sulfur-iodine cycle, copper-chlorinecycle, calcium-bromine-iron cycle, hybrid sulfur cycle; electrolysis ofhydrogen sulfide; thermochemical and/or electrochemical splitting ofhydrogen sulfide; other electrochemical or thermochemical processesknown to produce hydrogen with low- or no-carbon dioxide emissionsincluding but not limited to: carbon capture and sequestration enabledmethane reforming; carbon capture and sequestration enabled coalgasification; the Kværner-process and other processes generating acarbon-black product; carbon capture and sequestration enabledgasification or pyrolysis of biomass; and the half-cell reduction of H⁺to H₂ accompanied by the half-cell oxidization of electron sourcesincluding but not limited to ferrous iron (Fe²⁺) oxidized to ferric iron(Fe³⁺) or the oxidation of sulfur compounds whereby the oxidized iron orsulfur can be recycled to back to a reduced state through additionalchemical reaction with minerals including but not limited to metalsulfides, hydrogen sulfide, or hydrocarbons.

In certain embodiment of the present invention, hydrogen electron donorsare not necessarily generated with low- or no-carbon dioxide emissions,however the hydrogen is generated from waste or low value sources ofenergy using methods known in to art of chemical and process engineeringincluding but not limited to gasification, pyrolysis, or steam-reformingof feedstock such as but not limited to municipal waste, black liquor,agricultural waste, wood waste, stranded natural gas, biogas, sour gas,methane hydrates, tires, sewage, manure, straw, and low value, highlylignocellulosic biomass in general.

In certain embodiments of the present invention that utilize molecularhydrogen as an electron donor for the carbon-fixation reactionsperformed by oxyhydrogen microorganisms, there can be a chemicalco-product formed in the generation of molecular hydrogen using arenewable and/or CO₂ emission-free energy input. If water is used as ahydrogen source, then oxygen can be a co-product of water splittingthrough processes including but not limited to electrolysis orthermochemical water splitting. In certain embodiments of the presentinvention using water as a hydrogen source, some of the oxygenco-product can be used in the oxyhydrogen carbon fixation step for theproduction of intracellular ATP through the oxyhydrogen reactionenzymatically linked to oxidative phosphorylation. In certainembodiments of the present invention, the oxygen produced bywater-splitting in excess of what is required to maintain favorable(e.g., optimal) conditions for carbon fixation and organic compoundproduction by the oxyhydrogen microorganisms can be processed into aform suitable for sale through process steps known in the art andscience of commercial oxygen gas production. In certain embodiments ofthe present invention where hydrogen sulfide is the hydrogen source,sulfur or sulfuric acid can be a chemical co-product of molecularhydrogen production. In certain embodiments of the present inventionwhere sulfuric acid is a co-product of hydrogen production, some of thesulfuric acid can be used in the hydrolysis of biomass in post-carbonfixation process steps. In certain embodiments of the present invention,excess sulfuric acid and/or sulfur that is co-produced (e.g., beyondwhat can be used elsewhere in the carbon capture and conversion processof certain embodiments of the present invention) can be processed into aform suitable for sale through process steps known in the art andscience of commercial sulfuric acid and/or sulfur production. Processheat can also be generated in the production of hydrogen from hydrogensulfide. In certain embodiments of the present invention, process heatgenerated in hydrogen production is recovered and utilized elsewhere inthe carbon capture and conversion process of certain embodiments of thepresent invention to improve overall energy efficiency. A chemicaland/or heat and/or electrical co-product can accompany the generation ofmolecular hydrogen for use as an electron donor in certain embodimentsof the present invention. The chemical and/or heat and/or electricalco-products of molecular hydrogen generation can be used to the extentpossible elsewhere in the carbon capture and conversion process ofcertain embodiments of the present invention, for example, in order toimprove efficiency In certain embodiments, additional chemicalco-product (e.g., beyond what can be used in the carbon capture andconversion process of certain embodiments of the present invention) canbe prepared for sale in order to generate an additional stream ofrevenue. Excess heat or electrical energy co-product in the productionof molecular hydrogen (e.g., beyond what can be used internally in theprocess) can be delivered for sale, for example, for use in anotherchemical and/or biological process through means known in the art andscience heat exchange and transfer and electrical generation andtransmission, including but not limited to the conversion of processheat to electrical power in a form that can be sold.

Certain embodiments of the present invention utilize electrochemicalenergy stored in solid-state valence or conduction electrons within anelectrode or capacitor or related devices, alone or in combination withchemical electron donors and/or electron mediators to provide theoxyhydrogen microorganisms reducing equivalents for the carbon-fixationreactions by means of direct exposure of said electrode materials to themicrobial culturing environment and/or immersion of said electrodematerials within the microbial culture medium.

A feature of certain embodiments of the present invention regards theproduction, or recycling of electron donors generated from mineralogicalorigin that may also be used by certain oxyhydrogen microbes as a sourceof reducing equivalents in addition, or in lieu of hydrogen, includingbut not limited to electron donors generated from reduced S and Fecontaining minerals. Hence the present invention, in certainembodiments, can enable the use of a largely untapped source ofenergy—inorganic geochemical energy.

The electron donors used in certain embodiments of the present inventionmay be refined from natural mineralogical sources which include but arenot limited to one or more of the following: elemental Fe⁰; siderite(FeCO₃); magnetite (Fe₃O₄); pyrite or marcasite (FeS₂), pyrrhotite(Fe_((1-x))S (x=0 to 0.2)), pentlandite (Fe,Ni)₉S₈, violarite (Ni₂FeS₄),bravoite (Ni,Fe)S₂, arsenopyrite (FeAsS), or other iron sulfides;realgar (AsS); orpiment (As₂S₃); cobaltite (CoAsS); rhodochrosite(MnCO₃); chalcopyrite (CuFeS₂), bornite (Cu₅FeS₄), covellite (CuS),tetrahedrite (Cu₈Sb₂S₇), enargite (Cu₃AsS₄), tennantite (Cu₁₂As₄.S₁₃),chalcocite (Cu₂S), or other copper sulfides; sphalerite (ZnS), marmatite(ZnS), or other zinc sulfides; galena (PbS), geocronite (Pb₅(Sb,As₂)S₈),or other lead sulfides; argentite or acanthite (Ag₂S); molybdenite(MoS₂); millerite (NiS), polydymite (Ni₃S₄) or other nickel sulfides;antimonite (Sb₂S₃); Ga₂S₃; CuSe; cooperite (PtS); laurite (RuS₂);braggite (Pt,Pd,Ni)S; FeCl₂.

The generation of electron donor from natural mineralogical sourcesincludes a preprocessing step in certain embodiments of the presentinvention which can include but is not limited to comminuting, crushingor grinding mineral ore to increase the surface area for leaching withequipment such as a ball mill and wetting the mineral ore to make aslurry. In these embodiments of the present invention where electrondonors are generated from natural mineral sources, it may beadvantageous if particle size is controlled so that the sulfide and/orother reducing agents present in the ore may be concentrated by methodsknown to the art including but not limited to: flotation methods such asdissolved air flotation or froth flotation using flotation columns ormechanical flotation cells; gravity separation; magnetic separation;heavy media separation; selective agglomeration; water separation; orfractional distillation. After the production of crushed ore or slurry,the particulate matter in the leachate or concentrate may be separatedby filtering (e.g. vacuum filtering), settling, or other well knowntechniques of solid/liquid separation, prior to introducing the electrondonor containing solution to the chemoautotrophic culture environment.In addition anything toxic to the chemoautotrophs that is leached fromthe mineral ore may be removed prior to exposing the chemoautotrophs tothe leachate. The solid left after processing the mineral ore may beconcentrated with a filter press, disposed of, retained for furtherprocessing, or sold depending upon the mineral ore used in theparticular embodiment of the invention.

The electron donors in certain embodiments of the present invention mayalso be refined from pollutants or waste products including but notlimited to one or more of the following: process gas; tail gas; enhancedoil recovery vent gas; biogas; acid mine drainage; landfill leachate;landfill gas; geothermal gas; geothermal sludge or brine; metalcontaminants; gangue; tailings; sulfides; disulfides; mercaptansincluding but not limited to methyl and dimethyl mercaptan, ethylmercaptan; carbonyl sulfide; carbon disulfide; alkanesulfonates; dialkylsulfides; thiosulfate; thiofurans; thiocyanates; isothiocyanates;thioureas; thiols; thiophenols; thioethers; thiophene; dibenzothiophene;tetrathionate; dithionite; thionate; dialkyl disulfides; sulfones;sulfoxides; sulfolanes; sulfonic acid; dimethylsulfoniopropionate;sulfonic esters; hydrogen sulfide; sulfate esters; organic sulfur;sulfur dioxide and all other sour gases.

In addition to mineralogical sources, electron donors are produced orrecycled in certain embodiments of the present invention throughchemical reactions with hydrocarbons that may be of fossil origin, butwhich are used in chemical reactions producing low or zero carbondioxide gas emissions. These reactions include thermochemical andelectrochemical processes. Such chemical reactions that are used inthese embodiments of the present invention include but are not limitedto: the thermochemical reduction of sulfate reaction or TSR and theMuller-Kuhne reaction; methane reforming-like reactions utilizing metaloxides in place of water such as but not limited to iron oxide, calciumoxide, or magnesium oxide whereby the hydrocarbon is reacted to formsolid carbonate with little or no emissions of carbon dioxide gas alongwith hydrogen electron donor product.

Examples of reactions between metal oxides and hydrocarbons to produce ahydrogen electron donor product and carbonates include but are notlimited to:

2CH₄+Fe₂O₃+3H₂O→2FeCO₃+7H₂

and/or

CH₄+CaO+2H₂O→CaCO₃+4H₂.

In certain embodiments, the generated electron donors are oxidized inthe chemosynthetic reaction step or steps by electron acceptors thatinclude but are not limited to carbon dioxide, oxygen and/or one or moreof the following: ferric iron or other transition metal ions, nitrates,nitrites, sulfates, or valence or conduction band holes in solid stateelectrode materials.

The position of the chemosynthetic and/or oxyhydrogen reaction step orsteps in the general process flow of certain embodiments of the presentinvention is illustrated in FIG. 1 by Box 4 labeled “Bioreactor—KnallgasMicrobes.”

At each step in the process where chemosynthetic and/or oxyhydrogenreactions occur one or more types of electron donor and one or moretypes of electron acceptor may be pumped or otherwise added to thereaction vessel as either a bolus addition, or periodically, orcontinuously to the nutrient medium containing oxyhydrogenmicroorganisms. The chemosynthetic reaction driven by the transfer ofelectrons from electron donor to electron acceptor can fix inorganiccarbon dioxide into organic compounds and biomass.

In certain embodiments of the present invention electron mediators maybe included in the nutrient medium to facilitate the delivery ofreducing equivalents from electron donors to oxyhydrogen organisms inthe presence of electron acceptors and inorganic carbon in order tokinetically enhance the chemosynthetic reaction step. This aspect of thepresent invention can be used to enhance the transfer of reducingelectrons to the oxyhydrogen microbes from poorly soluble electrondonors such as but not limited to H₂ gas or electrons in solid stateelectrode materials using electron mediators known in the art ofelectrical stimulation of microbial metabolism including but not limitedto anthroquinone-2,6-disulfonate (AQDS), cobalt sepulchrate,cytochromes, formate, humic substances, iron, methyl-viologen,NAD+/NADH, neutral red (NR), phenazines, and quinones.

The delivery of reducing equivalents from electron donors to theoxyhydrogen microorganisms for the chemosynthetic reaction or reactionscan be kinetically and/or thermodynamically enhanced in certainembodiments through means including but not limited to: the introductionof hydrogen storage materials into the microbial culture environmentthat can double as a solid support media for microbial growth—bringingabsorbed or adsorbed hydrogen electron donors into close proximity withthe hydrogen-oxidizing chemoautotrophs and/or the introduction ofelectrode materials (e.g., graphite, graphite felt, activated carbon,carbon nanofibers, conductive polymers, steel, iron, copper, titanium,lead, tin, palladium, platinum, platinum-coated titanium, other platinumcoated metals, transition metals, transition metal alloys, transitionmetal sulfides, oxides, chalcogenides, halides, hydroxides,oxyhydroxides, phosphates, sulfates, and/or carbonates) that can doubleas a solid growth support media and a source of electron donors oracceptors directly into the chemoautotrophic cultureenvironment—bringing solid state electrons into close proximity with themicrobes. Some such embodiments of the present invention can be usefulfor transferring reducing equivalents from poorly soluble electrondonors such as but not limited to H₂ gas or electrons in solid stateelectrode materials to the oxyhydrogen microorganisms.

The culture broth used in the chemosynthetic steps of certainembodiments of the present invention may be an aqueous solutioncontaining suitable minerals, salts, vitamins, cofactors, buffers, andother components needed for microbial growth, known to those skilled inthe art [Bailey and Ollis, Biochemical Engineering Fundamentals, 2nd ed;pp 383-384 and 620-622; McGraw-Hill: New York (1986)]. These nutrientscan be chosen to maximize carbon-fixation and promote the carbon flowthrough enzymatic pathways leading to desired organic compounds.Alternative growth environments such as those used in the arts of solidstate or non-aqueous fermentation may be used in certain embodiments. Incertain embodiments that utilize an aqueous culture, broth, salt water,sea water and/or water from other natural bodies of water, or othernon-potable sources of water may be used when tolerated by theoxyhydrogen microorganisms.

The biochemical pathways may be controlled and optimized in certainembodiments of the present invention for the production of chemicalproducts (e.g., targeted organic compounds) and/or biomass bymaintaining specific growth conditions (e.g., levels of nitrogen,oxygen, phosphorous, sulfur, trace micronutrients such as inorganicions, and if present any regulatory molecules that might not generallybe considered a nutrient or energy source). Depending upon theembodiment of the invention the broth may be maintained in aerobic,microaerobic, anoxic, anaerobic, or facultative conditions. Afacultative environment is considered to be one having aerobic upperlayers and anaerobic lower layers caused by stratification of the watercolumn

The oxygen level is controlled in certain embodiments of the invention.The oxygen level can be controlled, for example, to enhance theproduction of targeted organic compounds by the oxyhydrogenmicroorganisms through carbon-fixation. One objective of controllingoxygen levels, in certain embodiments, is to control (e.g., optimize)the intracellular Adenosine Triphosphate (ATP) concentration through thecellular reduction of oxygen and production of ATP by oxidativephosphorylation. In some such embodiments, it can be desirable, whilecontrolling ATP concentration, to simultaneously keep the environmentsufficiently reducing so that the intracellular ratio of NADH (or NADPH)to NAD (or NADP) remains relatively high. In some embodiments, ATPlevels are increased and/or optimized within the oxyhydrogenmicroorganisms by means including but not limited to one or more of thefollowing: the cellular reduction of oxygen and/or another electronacceptor of sufficient oxidation strength for ATP production throughoxidative phosphorylation; the direct introduction of ATP into theculture medium; and/or the direct introduction of chemical analogues ofATP into the culture medium.

The reduction of oxygen by hydrogen in the oxyhydrogen reaction isgenerally enzymatically linked to the production of ATP throughoxidative phosphorylation in oxyhydrogen microorganisms. The oxyhydrogenreaction can act as a proxy for the light reaction in photosynthesis ingenerating both NADPH and ATP. Generally, in oxyhydrogen microorganisms,hydrogenase catalyzes the reduction of NAD to NADH by hydrogen (or,alternatively, in some photosynthetic organisms that are capable ofcarrying out the oxhydrogen reaction, a hydrogenase catalyzes thereduction of ferrodoxin by H₂, which in turn reduces NADP to NADPH)[Chen, Gibbs, Plant Physiol. (1992) 100, 1361-1365]. NADH and/or NADPHcan then be used as reducing agents for anabolic reactions, or togenerate ATP by reducing oxygen through oxidative phosphorylation[Bongers, J. Bacteriology, (October 1970) 145-151]. Therefore, in placeof the following light dependent photosynthetic reaction:

2H₂O+2NADP⁺+2ADP+2Pi+light→2NADPH +2H⁺+2ATP+O₂

an oxyhydrogen reaction of

1/2O₂+2NADP⁺+2ADP+2Pi+3H₂→2NADPH+2H⁺+2ATP+2H₂O

can occur in dark conditions (e.g., in the substantial absence ofvisible electromagnetic radiation), with hydrogen acting in the place ofphotons given the production of 2ATP per H₂ consumed [Bongers, J.Bacteriology, (October 1970) 145-151].

The maintenance of high intracellular concentrations of ATP as well asNADH and/or NADPH is targeted in certain embodiments of the presentinvention to promote carbon fixation and drive anabolic pathways and/orsolventogenic pathways that consume reducing equivalents and eitherconsume ATP, and/or that lower the net ATP yield of chemosyntheticcarbon-fixation. Such biochemical pathways include but are not limitedto the following: fatty acid synthesis; mevalonate pathway and terpenoidsynthesis; butanol pathway and 1-butanol synthesis;acetolactate/alpha-ketovalerate pathway and 2-butanol synthesis; and theethanol pathway. A preferred oxygen level can be determined, in someembodiments of in the present invention: too low an oxygen level canreduce the intracellular ATP in oxyhydrogen microorganisms below adesired level, while too high an oxygen level can decrease the NADH (orNADPH) to NAD (or NADP) ratio below a desired level.

The application of the oxyhydrogen reaction for the production of ATPand NADH and/or NADPH used for carbon fixation and synthesis of organiccompounds in certain embodiments of the present invention can provideadvantages over alternative approaches using, for example, anaerobicbiochemical pathways for carbon-fixation for such as Wood-Ljungdahl ormethanogenic pathways. Carbon-fixation through the Wood-Ljungdahl ormethanogenic pathways generally produces C1 or C2 organic compounds andit can be difficult to produce longer than C4 compounds through thesepathways.

The Wood-Ljungdahl pathway can produce acetic acid, ethanol, butyricacid, and butanol in nature, but butyric acid and butanol are generallyminor products of H₂ and CO₂ gas fermentation, and chain lengths longerthan C4 do not typically arise [Lynd, Zeikus, J. of Bacteriology (1983)1415-1423; Eichler, Schink, Archives of Microbiology (1984) 140,147-152]. The acetogenic pathways to acetic acid and butyric acidproduce net ATP, while the solventogenic pathways to ethanol and butanoldo not [Papoutsakis, Biotechnology & Bioengineering (1984) 26, 174-187;Heise, Muller, Gottschalk, J. of Bacteriology (1989) 5473-5478; Lee,Park, Jang, Nielsen, Kim, Jung, Biotechnology & Bioengineering (2008)101, 2, 209-228]. Since ATP is needed for cell maintenance a certainamount of relatively undesirable non-biofuel co-product (ie organicacids) from acetogens fixing carbon through the Wood-Ljungdahl pathwaywill generally be present which constitutes a waste of reducingequivalents and carbon.

The production of hydrocarbons with chain length longer than C4 is mostcommonly accomplished biologically through fatty acid biosynthesis[Fischer, Klein-Marcuschamer, Stephanolpoulos, Metabolic Engineering(2008) 10, 295-304]. Unlike the solventogenic pathways coming out of theWood-Ljungdahl pathway, fatty acid synthesis involves net ATPconsumption. For example the following gives the net reaction forsynthesis of Palmitic acid (C16), in this example starting fromAcetyl-CoA: 8Acetyl-CoA+7ATP+H2O+14NADPH+14H⁺->Palmiticacid+8CoA+14NADP⁺+7ADP+7Pi

One difficulty with using anaerobic pathways such as Methanogenesis orWood-Ljungdahl for ATP production to drive fatty acid synthesis is theATP produced per H₂ consumed is relatively low: one ATP per 4H₂ formethane [Thauer, R. K., Kaster, A. K., Seedorf, H., Buckel, W. &Hedderich, R. Methanogenic archaea: ecologically relevant differences inenergy conservation. Nat Rev Microbiol 6, 579-591, doi:nrmicro1931[pii]] or acetic acid production and one ATP per 10H₂ for butyric acidproduction [Papoutsakis, Biotechnology & Bioengineering (1984) 26,174-187; Heise, Muller, Gottschalk, J. of Bacteriology (1989) 5473-5478;Lee, Park, Jang, Nielsen, Kim, Jung, Biotechnology & Bioengineering(2008) 101, 2, 209-228]. By contrast, for the oxyhydrogen reaction,hydrogenotrophic oxyhydrogen microorganisms can produce up to two ATPper H₂ consumed [Bongers, J. Bacteriology, (October 1970) 145-151]. Inother words oxyhydrogen microorganisms can produce up to eight timesmore ATP per H₂ consumed than methanogenic or acetogenic microorganisms.Furthermore the path to ATP production through the oxyhydrogen reactionproduces water which can readily be incorporated into the process streamrather than the relatively undesirable acetic acid or butyric acidproducts of acidogenesis that can upset the system pH and can rise toconcentrations toxic to the organisms.

The highest energy density fuel that can be practically reachednaturally through the Wood-Ljungdahl pathway with inorganic carbon inputis generally ethanol at 30 MJ/kg, although butanol at 36.1 MJ/kg mightbe possible. Production of diesel fuels (46.2 MJ/kg) or JP-8 aviationfuel (43.15 MJ/kg) can generally be difficult and is generally lessefficient utilizing anaerobic pathways such as Wood-Ljungdahl due to theincreased amount of H₂ that needs to be consumed in strictly anaerobicpathways per ATP produced, which is needed for fatty acid synthesis.However these high density, infrastructure compatible liquid fuels canbe readily produced through fatty synthesis pathways driven by ATP andNADH or NADPH generated by the oxyhydrogen reaction.

Biomass lipid content and lipid biosynthetic pathway efficiency are twofactors that can affect the overall efficiency of certain embodiments ofthe present invention for converting CO₂ and other C1 compounds tolonger chain compounds (e.g., infrastructure-compatible fuels). Thebiomass lipid content can determine the proportion of carbon andreducing equivalents directed towards the synthesis of fuel products, asopposed to other components of biomass. The lipid content can determinethe amount of energy input from the reducing equivalents that can becaptured in final fuel product. Likewise, the metabolic pathwayefficiency can determine the amount of reducing equivalents that must beconsumed in converting CO₂ and hydrogen to lipid along the lipidbiosynthetic pathway. Many oxyhydrogen microorganisms include speciesrich in lipid content and containing efficient pathways from H₂ and CO₂to lipid. Certain embodiments of the present invention use species withhigh lipid contents such as but not limited to Rhodococcus opacus whichcan have a lipid content of over 70% [Gouda, M. K., Omar, S. H.,Chekroud, Z. A. & Nour Eldin, H. M. Bioremediation of kerosene I: A casestudy in liquid media. Chemosphere 69, 1807-1814,doi:S0045-6535(07)00738-2; Waltermann, M., Luftmann, H., Baumeister, D.,Kalscheuer, R. & Steinbuchel, A. Rhodococcus opacus strain PD630 as anew source of high-value single-cell oil? Isolation and characterizationof triacylglycerols and other storage lipids. Microbiology 146 (Pt 5),1143-1149 (2000).] and/or species utilizing highly efficiency metabolicpathways such as but not limited to the reverse tricarboxylic acid cycle[i.e. reverse citric acid cycle] to fix carbon [Miura, A., Kameya, M.,Arai, H., Ishii, M. & Igarashi, Y. A soluble NADH-dependent fumaratereductase in the reductive tricarboxylic acid cycle of Hydrogenobacterthermophilus TK-6. J Bacteriol 190, 7170-7177, doi:JB.00747-08 [pii]10.1128/JB.00747-08 (2008).; Shively, J. M., van Keulen, G. & Meijer, W.G. Something from almost nothing: carbon dioxide fixation inchemoautotrophs. Annu Rev Microbiol 52, 191-230,doi:10.1146/annurev.micro.52.1.191 (1998).]. In terms of energyefficiency, the reverse tricarboxylic acid pathway can be a relativelyfavorable pathway. The synthesis of palmitic acid from H₂ and CO₂ isgenerally about 15% more efficient in terms of reducing equivalentsconsumed than palmitic acid synthesis in acetogens, due to the increasedATP output per reducing equivalent consumed in the oxyhydrogen reactionby oxyhydrogen microorganisms.

The source of inorganic carbon used in the chemosynthetic reactionprocess steps of certain embodiments of the present invention includesbut is not limited to one or more of the following: a carbondioxide-containing gas stream that may be pure or a mixture; liquefiedCO₂; dry ice; dissolved carbon dioxide, carbonate ion, or bicarbonateion in solutions including aqueous solutions such as sea water;inorganic carbon in a solid form such as a carbonate or bicarbonateminerals. Carbon dioxide and/or other forms of inorganic carbon can beintroduced to the nutrient medium contained in reaction vessels eitheras a bolus addition, periodically, or continuously at the steps in theprocess where carbon-fixation occurs. Organic compounds containing onlyone carbon atom that can be used in the synthetic reaction process stepsof certain embodiments of the present invention include but are notlimited to one or more of the following: carbon monoxide, methane,methanol, formate, formic acid, and/or mixtures containing C1 chemicalsincluding but not limited to various syngas compositions generated fromvarious gasified or steam-reformed fixed carbon feedstocks.

In certain embodiments, organic compounds containing only one carbonatom and/or electron donors are generated through the gasificationand/or pyrolysis of biomass and/or other organic matter (e.g., biomassand/or other organic matter from waste or low value sources), andprovided as a syngas to the culture of oxyhydrogen microorganism, wherethe ratio of hydrogen to carbon monoxide in the syngas may or may not beadjusted through means such as the water gas shift reaction, prior tothe syngas being delivered to the microbial culture. In certainembodiments, organic compounds containing only one carbon atom and/orelectron donors are generated through methane steam reforming frommethane or natural gas (e.g., stranded natural gas, or natural gas thatwould be otherwise flared or released to the atmosphere), or biogas, orlandfill gas, and provided as a syngas to the culture of oxyhydrogenmicroorganism, where the ratio of hydrogen to carbon monoxide in thesyngas may or may not be adjusted through means such as the water gasshift reaction, prior to the syngas being delivered to the microbialculture.

In certain embodiments of the present invention, carbon dioxidecontaining flue gases are captured from the smoke stack at temperature,pressure, and gas composition characteristic of the untreated exhaust,and directed with minimal modification into the reaction vessels wherecarbon-fixation occurs. In some embodiments in which impurities harmfulto chemoautotrophic organisms are not present in the flue gas,modification of the flue gas upon entering the reaction vessels can belimited to the compression needed to pump the gas through the reactorsystem and/or the heat exchange needed to lower the gas temperature toone suitable for the microorganisms.

Oxyhydrogen microorganisms generally have an advantage over strictanaerobic acetogenic or methanogenic microorganisms for carbon captureapplications due to the higher oxygen tolerance of oxyhydrogenmicroorganisms. Since industrial flue gas is one intended source of CO₂for certain embodiments of the present invention, the relatively highoxygen tolerance of oxyhydrogen microorganisms, as compared withobligately anaerobic methanogens or acetogens, can allow the O₂ contentof 2-6% found in typical fluegas to be tolerated.

In embodiments in which carbon dioxide bearing flue gas is transportedthrough a system for dissolving the carbon dioxide into solution (suchas is well known in the art of carbon capture), the scrubbed flue gas,(which generally primarily includes inert gases such as nitrogen), canbe released into the atmosphere.

Gases in addition to carbon dioxide that are dissolved into solution andfed to the culture broth or dissolved directly into the culture broth incertain embodiments of the present invention include gaseous electrondonors (e.g., hydrogen gas), but in certain embodiments of the presentinvention, may include other electron donors such as but not limited tocarbon monoxide and other constituents of syngas, hydrogen sulfide,and/or other sour gases. A controlled amount of oxygen can also bemaintained in the culture broth of some embodiments of the presentinvention, and in certain embodiments, oxygen will be actively dissolvedinto solution fed to the culture broth and/or directly dissolved intothe culture broth.

The dissolution of oxygen, carbon dioxide, and/or electron donor gasessuch as but not limited to hydrogen and/or carbon monoxide into solutioncan be achieved in some embodiments of the present invention using asystem of compressors, flowmeters, and/or flow valves known to oneskilled in the art of bioreactor scale microbial culturing, which can befed into one of more of the following commonly used systems for pumpinggas into solution: sparging equipment; diffusers including but notlimited to dome, tubular, disc, or doughnut geometries; coarse or finebubble aerators; and/or venturi equipment. In certain embodiments of thepresent invention, surface aeration may also be performed using paddleaerators and the like. In certain embodiments of the present invention,gas dissolution is enhanced by mechanical mixing with an impeller and/orturbine. In some embodiments, hydraulic shear devices can be used toreduce bubble size.

In certain embodiments of the present invention that require the activepumping of air or oxygen into the culture broth in order to maintainfavorable (e.g., optimal) oxygenation levels, oxygen bubbles areinjected into the broth at a desirable (e.g., the optimal) diameter formixing and oxygen transfer. This has been found to be 2 mm for certainembodiments [Environment Research Journal May/June 1999 pgs. 307-315].In certain aerobic embodiments of the present invention, a process ofshearing the oxygen bubbles is used to achieve this bubble diameter asdescribed in U.S. Pat. No. 7,332,077. In some embodiments, bubbles havean average diameter of no larger than 7.5 mm and slugging is avoided.

In certain embodiments of the present invention utilizing hydrogen aselectron donor, hydrogen gas is fed to the chemoautotrophic culturevessel by bubbling it through the culture medium and/or by diffusing itthrough a membrane that contacts the culture medium and is impermeableto the culture medium. The latter method is considered safer for manyembodiments, and can be preferred since hydrogen accumulating in the gasphase can create explosive conditions (the range of explosive hydrogenconcentrations in air is 4 to 74.5% and can be avoided in certainembodiments of the present invention). In some embodiments, the membraneis coated with a biofilm of the oxyhydrogen microorganisms such that thehydrogen must diffuse through the microorganism after passage throughthe membrane.

Additional chemicals required or useful for the maintenance and growthof oxyhydrogen microorganisms as known in the art can be added to theculture broth of certain embodiments of the present invention. Thesechemicals may include but are not limited to: nitrogen sources such asammonia, ammonium (e.g. ammonium chloride (NH₄Cl), ammonium sulfate((NH₄)₂SO₄)), nitrate (e.g. potassium nitrate (KNO₃)), urea or anorganic nitrogen source; phosphate (e.g. disodium phosphate (Na₂HPO₄),potassium phosphate (KH₂PO₄), phosphoric acid (H₃PO₄), potassiumdithiophosphate (K₃PS₂O₂), potassium orthophosphate (K₃PO₄), dipotassiumphosphate (K₂HPO₄)); sulfate; yeast extract; chelated iron; potassium(e.g. potassium phosphate (KH₂PO₄), potassium nitrate (KNO₃), potassiumiodide (KI), potassium bromide (KBr)); and other inorganic salts,minerals, and trace nutrients (e.g. sodium chloride (NaCl), magnesiumsulfate (MgSO₄ 7H₂O) or magnesium chloride (MgCl₂), calcium chloride(CaCl₂) or calcium carbonate (CaCO₃), manganese sulfate (MnSO₄ 7H₂O) ormanganese chloride (MnCl₂), ferric chloride (FeCl₃), ferrous sulfate(FeSO₄ 7H₂O) or ferrous chloride (FeCl₂ 4H₂O), sodium bicarbonate(NaHCO₃) or sodium carbonate (Na₂CO₃), zinc sulfate (ZnSO₄) or zincchloride (ZnCl₂), ammonium molybdate (NH₄MoO₄) or sodium molybdate(Na₂MoO₄ 2H₂O), cuprous sulfate (CuSO₄) or copper chloride (CuCl₂ 2H₂O),cobalt chloride (CoCl₂ 6H₂O), aluminum chloride (AlCl₃.6H₂O), lithiumchloride (LiCl), boric acid (H₃BO₃), nickel chloride NiCl₂ 6H₂O), tinchloride (SnCl₂ H₂O), barium chloride (BaCl₂ 2H₂O), copper selenate(CuSeO₄ 5H₂O) or sodium selenite (Na₂SeO₃), sodium metavanadate (NaVO₃),chromium salts). In certain embodiments the mineral salts medium (MSM)formulated by Schlegel et al may be used [Thermophilic bacteria, JakobKristjansson, Chapter 5, Section III, CRC Press, (1992)].

In certain embodiments, the concentrations of nutrient chemicals (e.g.,the electron donors and acceptors), are maintained at favorable levels(e.g., as close as possible to their respective optimal levels) forenhanced (e.g., maximum) carbon uptake and fixation and/or production oforganic compounds, which varies depending upon the oxyhydrogen speciesutilized but is known or determinable without undue experimentation toone of ordinary skill in the art of culturing oxyhydrogenmicroorganisms.

Along with nutrient levels, the waste product levels, pH, temperature,salinity, dissolved oxygen and carbon dioxide, gas and liquid flowrates, agitation rate, and pressure in the microbial culture environmentare controlled in certain embodiments of the present invention. Theoperating parameters affecting carbon-fixation can be monitored withsensors (e.g. using a dissolved oxygen probe and/or anoxidation-reduction probe to gauge electron donor/acceptorconcentrations) and can be controlled either manually or automaticallybased upon feedback from sensors through the use of equipment includingbut not limited to actuating valves, pumps, and agitators. Thetemperature of the incoming broth as well as incoming gases can beregulated by means such as but not limited to heat exchangers.

The dissolution of gases and nutrients needed to maintain theoxyhydrogen culture and promote carbon-fixation, as well as the removalof inhibitory waste products, can be enhanced by agitation of theculture broth. Oxyhydrogen microorganisms can carry out carbon-fixationreactions throughout the volume of the reaction vessel, which providesan advantage over other approaches including those that employphotosynthetic organisms, which are surface area limited due to thelight requirements of photosynthesis. The use of agitation can furtherenhance this advantage by distributing the microorganisms, nutrients,optimal growth environment, and/or CO₂ as widely and evenly as possiblethroughout the reactor volume so that production is enhanced (e.g., thereactor volume in which carbon-fixation reactions occur at an optimalrate is maximized).

Agitation of the culture broth in certain embodiments of the presentinvention can be accomplished by equipment including but not limited to:recirculation of broth from the bottom of the container to the top via arecirculation conduit; sparging with carbon dioxide, electron donor gas(e.g. H₂), oxygen, and/or air; and/or a mechanical mixer such as but notlimited to an impeller (100-1000 rpm) or turbine.

In certain embodiments of the present invention, the chemicalenvironment, oxyhydrogen microorganisms, electron donors, electronacceptors, oxygen, pH, and/or temperature levels are varied eitherspatially and/or temporally over a series of bioreactors in fluidcommunication, such that a number of different carbon-fixation reactionsand/or biochemical pathways to organic compounds are carried outsequentially or in parallel.

The nutrient medium containing oxyhydrogen microorganisms can be removedfrom the bioreactors in certain embodiments of the present inventionpartially or completely, periodically or continuously, and can bereplaced with fresh cell-free medium, for example, to maintain the cellculture in an exponential growth phase, to maintain the cell culture ina growth phase (exponential or stationary) with enhanced (e.g., optimal)carbon-fixation rates, to replenish the depleted nutrients in the growthmedium, and/or remove inhibitory waste products.

The high growth rate attainable by oxyhydrogen species can allow them tomatch or surpass the highest rates of carbon fixation and/or biomassproduction per standing unit biomass that can be achieved byphotosynthetic microbes. Consequently, in certain embodiments, surplusbiomass can be produced. Surplus growth of cell mass can be removed fromthe system to produce a biomass product. In some embodiments, surplusgrowth of cell mass can be removed from the system in order to maintaina desirable (e.g., an optimal) microbial population and cell density inthe microbial culture for continued high carbon capture and fixationrates.

Another advantage of certain embodiments of the present inventionrelates to the vessels used to contain the carbon-fixation reactionenvironment and culture in the carbon capture and fixation process.Exemplary culture vessels that can be used in some embodiments of thepresent invention to culture and grow the oxyhydrogen microorganisms forcarbon dioxide capture and fixation include those that are known tothose of ordinary skill in the art of large scale microbial culturing.Such culture vessels, which may be of natural or artificial origin,include but are not limited to: airlift reactors; biological scrubbercolumns; bioreactors; bubble columns; caverns; caves; cisterns;continuous stirred tank reactors; counter-current, upflow, expanded-bedreactors; digesters and in particular digester systems such as known inthe prior arts of sewage and waste water treatment or bioremediation;filters including but not limited to trickling filters, rotatingbiological contactor filters, rotating discs, soil filters; fluidizedbed reactors; gas lift fermenters; immobilized cell reactors; lagoons;membrane biofilm reactors; microbial fuel cells; mine shafts; pachucatanks; packed-bed reactors; plug-flow reactors; ponds; pools; quarries;reservoirs; static mixers; tanks; towers; trickle bed reactors; vats;vertical shaft bioreactors; and wells. The vessel base, siding, walls,lining, and/or top can be constructed out of one or more materialsincluding but not limited to bitumen, cement, ceramics, clay, concrete,epoxy, fiberglass, glass, macadam, plastics, sand, sealant, soil, steelsor other metals and their alloys, stone, tar, wood, and any combinationthereof. In certain embodiments of the present invention where theoxyhydrogen microorganisms either require a corrosive growth environmentand/or produce corrosive chemicals through the carbon-fixation reaction,corrosion resistant materials can be used to line the interior of thecontainer contacting the growth medium.

Since oxyhydrogen microorganisms do not require sunlight in order to fixCO₂, they can be used in carbon capture and fixation processes thatavoid many of the shortcomings that can be associated withphotosynthetically based carbon capture and conversion technologies. Forexample, the maintenance of chemosynthesis does not require shallow,wide ponds, nor bioreactors with high surface area to volume ratios andspecial features like solar collectors or transparent materials. Atechnology such as certain embodiments of the present invention usingoxyhydrogen microbes does not have the diurnal, geographical,meteorological, or seasonal constraints typically associated withphotosynthetically based systems.

Certain embodiments of the present invention minimize material costs byusing chemosynthetic vessel geometries having a low surface area tovolume ratio, such as but not limited to cubic, cylindrical shapes withmedium aspect ratio, ellipsoidal or “egg-shaped”, hemispherical, orspherical shapes, unless material costs are superseded by other designconsiderations (e.g. land footprint size). The ability to use compactreactor geometries can arise from the absence of a light requirement forchemosynthetic reactions, in contrast to photosynthetic technologieswhere the surface area to volume ratio must be large to providesufficient light exposure.

The oxyhydrogen microorganisms' lack of dependence on light also canallow plant designs with a much smaller footprint than thosetraditionally associated with photosynthetic approaches. For example, inscenarios where the plant footprint needs to be minimized due torestricted land availability, a long vertical shaft bioreactor systemcan be used for chemosynthetic carbon capture. A bioreactor of the longvertical shaft type is described, for example, in U.S. Pat. Nos.4,279,754, 5,645,726, 5,650,070, and 7,332,077.

Unless superseded by other considerations, certain embodiments of thepresent invention minimize vessel surfaces across which high losses ofwater, nutrients, and/or heat occur, and/or the introduction of invasivepredators into the reactor. The ability to minimize such surfaces canarise from the lack of light requirements for chemosynthesis.Photosynthetic based technologies generally are not able to minimizesuch surfaces since surfaces across which high losses of water,nutrients, and/or heat occur, as well as losses due to predation aregenerally the same surfaces across which the light energy necessary forphotosynthesis is transmitted.

The culture vessels of the present invention can, in some embodiments,use reactor designs known to those of ordinary skill in the art of largescale microbial culture to maintain an aerobic, microaerobic, anoxic,anaerobic, or facultative environment depending upon the embodiment ofthe present invention. For example, similar to the design of many sewagetreatment facilities, in certain embodiments of the present invention,tanks are arranged in a sequence, with serial forward fluidcommunication, where certain tanks are maintained in aerobic conditionsand others are maintained in anaerobic conditions, in order to performmultiple chemosynthetic, and in certain embodiments, heterotrophic,processing steps on the carbon dioxide waste stream.

In certain embodiments of the present invention, the oxyhydrogenmicroorganisms are immobilized within their growth environmentImmobilization of the microorganisms can be accomplished using any mediaknown in the art of microbial culturing to support colonization bymicroorganisms including but not limited to growing the microorganismson a matrix, mesh, or membrane made from any of a wide range of naturaland synthetic materials and polymers including but not limited to one ormore of the following: glass wool, clay, concrete, wood fiber, inorganicoxides such as ZrO₂, Sb₂O₃, or Al₂O₃, the organic polymer polysulfone,or open-pore polyurethane foam having high specific surface area. Themicroorganisms in certain embodiments of the present invention may alsobe grown on the surfaces of unattached objects distributed throughoutthe growth container as are known in the art of microbial culturing thatinclude but are not limited to one or more of the following: beads;sand; silicates; sepiolite; glass; ceramics; small diameter plasticdiscs, spheres, tubes, particles, or other shapes known in the art;shredded coconut hulls; ground corn cobs; activated charcoal; granulatedcoal; crushed coral; sponge balls; suspended media; bits of smalldiameter rubber (elastomeric) polyethylene tubing; hanging strings ofporous fabric, Berl saddles, Raschig rings. The materials used in themicrobial support media may include hydrogen storage and/or electrodematerials in order to enhance the transfer of reducing equivalents tothe oxyhydrogen microorganisms. The electrode materials that can be usedinclude but are not limited to one or more of the following: graphite,activated carbon, carbon nanofibers, conductive polymers, steel, iron,copper, titanium, lead, tin, palladium, platinum, transition metals,transition metal alloys, transition metal sulfides, oxides,chalcogenides, halides, hydroxides, oxyhydroxides, phosphates, sulfates,or carbonates. The hydrogen storage materials that may be used in thisapplication include but are not limited to titanium, graphite, activatedcarbon, carbon nanofibers, iron, copper, lead, tin, metal hydridesincluding but not limited to TiFeH₂, TiH₂, VH₂, ZrH₂, NiH, NbH₂, PdH,and polymers known in the art of hydrogen storage including but notlimited to Metal Organic Frameworks (MOF), and nanoporous polymericmaterials. In certain embodiments, the hydrogen storage material doesnot react strongly with water or have a strong or rapid effect on the pHof the culture medium.

Inoculation of the oxyhydrogen culture into the culture vessel can beperformed by methods including but not limited to transfer of culturefrom an existing oxyhydrogen culture inhabiting another carbon captureand fixation system of certain embodiments of the present inventionand/or incubation from a seed stock raised in an incubator. The seedstock of oxyhydrogen strains can be transported and stored in formsincluding but not limited to a powder, a liquid, a frozen form, or afreeze-dried form as well as any other suitable form, which may bereadily recognized by one skilled in the art. In certain embodiments inwhich a culture is established in a very large reactor, growth andestablishment of cultures can be performed in progressively largerintermediate scale containers prior to inoculation of the full scalevessel.

The position of the process step or steps for the separation of cellmass from the process stream in the general process flow of certainembodiments of the present invention is illustrated in FIG. 1 by Box 5,labeled “Cell Separation”.

Separation of cell mass from liquid suspension can be performed bymethods known in the art of microbial culturing [Examples of cell massharvesting techniques are given in International Patent Application No.WO08/00558, published Jan. 8, 1998; U.S. Pat. No. 5,807,722; U.S. Pat.No. 5,593,886 and U.S. Pat. No. 5,821,111.]including but not limited toone or more of the following: centrifugation; flocculation; flotation;filtration using a membranous, hollow fiber, spiral wound, or ceramicfilter system; vacuum filtration; tangential flow filtration;clarification; settling; hydrocyclone. In certain embodiments where thecell mass is immobilized on a matrix, it can be harvested by methodsincluding but not limited to gravity sedimentation or filtration, andseparated from the growth substrate by liquid shear forces.

In certain embodiments of the present invention, if an excess of cellmass has been removed from the culture, it can be recycled back into thecell culture as indicated by the process arrow labeled “Recycled CellMass” in FIG. 1., along with fresh broth such that sufficient biomass isretained in the chemosynthetic reaction step or steps. This can allowfor continued enhanced (e.g., optimal) autotrophic carbon-fixation andproduction of organic compounds. The cell mass recovered by theharvesting system can be recycled back into the culture vessel, forexample, using an airlift or geyser pump. In certain embodiments, thecell mass recycled back into the culture vessel is not exposed toflocculating agents, unless those agents are non-toxic to themicroorganisms.

In certain embodiments of the present invention, the microbial cultureand carbon-fixation reaction is maintained using continuous influx andremoval of nutrient medium and/or biomass, in steady state where thecell population and environmental parameters (e.g. cell density,chemical concentrations) are targeted at a constant (e.g., optimal)level over time. Cell densities can be monitored in certain embodimentsof the present invention by direct sampling, by a correlation of opticaldensity to cell density, and/or with a particle size analyzer. Thehydraulic and biomass retention times can be decoupled so as to allowindependent control of both the broth chemistry and the cell density.Dilution rates can be kept high enough so that the hydraulic retentiontime is relatively low compared to the biomass retention time, resultingin a highly replenished broth for cell growth. Dilution rates can be setat an optimal trade-off between culture broth replenishment, andincreased process costs from pumping, increased inputs, and otherdemands that rise with dilution rates.

To assist in the processing of the biomass product into biofuels orother useful products, the surplus microbial cells in certainembodiments of the invention can be broken open following the cellrecycling step using, for example, methods including but not limited toball milling, cavitation pressure, sonication, or mechanical shearing.

The harvested biomass in some embodiments can be dried in the processstep or steps of Box 7, labeled “Dryer” in the general process flow ofcertain embodiments of the present invention illustrated in FIG. 1.

Surplus biomass drying can be performed in certain embodiments of thepresent invention using technologies including but not limited tocentrifugation, drum drying, evaporation, freeze drying, heating, spraydrying, vacuum drying, and/or vacuum filtration. Heat waste from theindustrial source of flue gas can be used in drying the biomass, incertain embodiments. In addition, the chemosynthetic oxidation ofelectron donors is generally exothermic and generally produces wasteheat. In certain embodiments of the present invention waste heat can beused in drying the biomass.

In certain embodiments of the invention, the biomass is furtherprocessed following drying to aid the production of biofuels or otheruseful chemicals through the separation of the lipid content or othertargeted biochemicals from the microbial biomass. The separation of thelipids can be performed by using nonpolar solvents to extract the lipidssuch as, but not limited to, hexane, cyclohexane, ethyl ether, alcohol(isopropanol, ethanol, etc.), tributyl phosphate, supercritical carbondioxide, trioctylphosphine oxide, secondary and tertiary amines, orpropane. Other useful biochemicals can be extracted using solventsincluding but not limited to: chloroform, acetone, ethyl acetate, andtetrachloroethylene.

The extracted lipid content of the biomass can be processed usingmethods known in the art and science of biomass refining including butnot limited to one or more of the following—catalytic cracking andreforming; decarboxylation; hydrotreatment; isomerization—to producepetroleum and petrochemical replacements, including but not limited toone or more of the following: JP-8 jet fuel, diesel, gasoline, and otheralkanes, olefins and aromatics. In some embodiments, the extracted lipidcontent of the biomass can be converted to ester-based fuels, such asbiodiesel (fatty acid methyl ester or fatty acid ethyl ester), throughprocesses known in the art and science of biomass refining including butnot limited to transesterification and esterification.

The broth left over following the removal of cell mass can be pumped toa system for removal of the chemical products of chemosynthesis and/orspent nutrients which are recycled or recovered to the extent possibleand/or disposed of.

The position of the process step or steps for the recovery of chemicalproducts from the process stream in the general process flow of certainembodiments of the present invention is illustrated in FIG. 1 by Box 8,labeled “Separation of chemical co-products.”

Recovery and/or recycling of chemosynthetic chemical products and/orspent nutrients from the aqueous broth solution can be accomplished incertain embodiments of the present invention using equipment andtechniques known in the art of process engineering, and targeted towardsthe chemical products of particular embodiments of the presentinvention, including but not limited to: solvent extraction; waterextraction; distillation; fractional distillation; cementation; chemicalprecipitation; alkaline solution absorption; absorption or adsorption onactivated carbon, ion-exchange resin or molecular sieve; modification ofthe solution pH and/or oxidation-reduction potential, evaporators,fractional crystallizers, solid/liquid separators, nanofiltration, andall combinations thereof.

In certain embodiments of the present invention, free fatty acids,lipids, or other medium or long chain organic compounds appropriate forrefinement to biofuel products that have been produced throughchemosynthesis can be recovered from the process stream at the step atBox 8 in FIG. 1. These free organic molecules can be released into theprocess stream solution from the oxyhydrogen microorganisms throughmeans including but not limited to cellular excretion or secretion orcell lysis. In certain embodiments of the present invention, therecovered organic compounds are processed using methods known in the artand science of biomass refining including but not limited to one or moreof the following: catalytic cracking and reforming; decarboxylation;hydrotreatment; isomerization. Such processes can be used to producepetroleum and petrochemical replacements, including but not limited toone or more of the following: JP-8 jet fuel, diesel, gasoline, and otheralkanes, olefins and aromatics. Recovered fatty acids can be convertedto ester-based fuels, such as biodiesel (fatty acid methyl ester orfatty acid ethyl ester), through processes known in the art and scienceof biomass refining including but not limited to transesterification andesterification.

In some embodiments, following the recovery of chemical products fromthe process stream, the removal of the waste products is performed asindicated by Box 9, labeled “Waste removal” in FIG. 1. The remainingbroth can be returned to the culture vessel along with replacement waterand/or nutrients.

In certain embodiments of the present invention involvingchemoautotrophic oxidization of electron donors extracted from a mineralore, a solution of oxidized metal cations can remain following thechemosynthetic reaction steps. A solution rich in dissolved metalcations can also result from a particularly dirty flue gas input to theprocess such as from a coal fired plant. In some such embodiments of thepresent invention, the process stream can be stripped of metal cationsby methods including but not limited to: cementation on scrap iron,steel wool, copper or zinc dust; chemical precipitation as a sulfide orhydroxide precipitate; electrowinning to plate a specific metal;absorption on activated carbon or an ion-exchange resin, modification ofthe solution pH and/or oxidation-reduction potential, solventextraction. In certain embodiments of the present invention, therecovered metals can be sold for an additional stream of revenue.

In certain embodiments, the chemicals that are used in processes for therecovery of chemical products, the recycling of nutrients and water, andthe removal of waste have low toxicity for humans, and if exposed to theprocess stream that is recycled back into the growth container, lowtoxicity for the oxyhydrogen microorganisms being used.

In certain embodiments of the present invention, the pH of the microbialculture is controlled. To address a decrease in pH, a neutralizationstep can be performed prior to recycling the broth back into the culturevessel in order to maintain the pH within an optimal range for microbialmaintenance and growth. Neutralization of acid in the broth can beaccomplished by the addition of bases including but not limited to:limestone, lime, sodium hydroxide, ammonia, caustic potash, magnesiumoxide, iron oxide. In certain embodiments, the base is produced from acarbon dioxide emission-free source such as naturally occurring basicminerals including but not limited to calcium oxide, magnesium oxide,iron oxide, iron ore, olivine containing a metal oxide, serpentinecontaining a metal oxide, ultramafic deposits containing metal oxides,and underground basic saline aquifers. If limestone is used forneutralization, then carbon dioxide will generally be released, whichcan be directed back into the growth container for uptake bychemosynthesis and/or sequestered in some other way, rather thanreleased into the atmosphere.

An additional feature of certain embodiments of the present inventionrelates to the uses of organic compounds and/or biomass produced throughthe chemosynthetic process step or steps of certain embodiments of thepresent invention. Uses of the organic compounds and/or biomass producedinclude but are not limited to: the production of liquid fuels includingbut not limited to JP-8 jet fuel, diesel, gasoline, octane, biodiesel,butanol, ethanol, propanol, isopropanol, propane, alkanes, olefins,aromatics, fatty alcohols, fatty acid esters, alcohols; the productionof organic chemicals including but not limited to 1,3-propanediol,1,3-butadiene, 1,4-butanediol, 3-hydroxypropionate,7-ADCA/cephalosporin, ε-caprolactone, γ-valerolactone, acrylate, acrylicacid, adipic acid, ascorbate, aspartate, ascorbic acid, aspartic acid,caprolactam, carotenoids, citrate, citric acid, DHA, docetaxel,erythromycin, ethylene, gamma butyrolactone, glutamate, glutamic acid,HPA, hydroxybutyrate, isopentenol, isoprene, isoprenoids, itaconate,itaconic acid, lactate, lactic acid, lanosterol, levulinic acid,lycopene, lysine, malate, malonic acid, peptides, omega-3 DHA, omegafatty acids, paclitaxel, PHA, PHB, polyketides, polyols, propylene,pyrrolidones, serine, sorbitol, statins, steroids, succinate,terephthalate, terpenes, THF, rubber, wax esters, polymers, commoditychemicals, industrial chemicals, specialty chemicals, paraffinreplacements, additives, nutritional supplements, neutraceuticals,pharmaceuticals, pharmaceutical intermediates, personal care products;as raw material and/or feedstock for manufacturing or chemicalprocesses; as feed stock for alcohol or other biofuel fermentationand/or gasification and liquefaction processes and/or other biofuelproduction processes including but not limited to catalytic cracking,direct liquefaction, Fisher Tropsch processes, hydrogenation, methanolsynthesis, pyrolysis, transesterification, or microbial syngasconversions; as a biomass fuel for combustion in particular as a fuel tobe co-fired with fossil fuels; as sources of pharmaceutical, medicinalor nutritional substances; as a carbon source for large scalefermentations to produce various chemicals including but not limited tocommercial enzymes, antibiotics, amino acids, vitamins, bioplastics,glycerol, or 1,3-propanediol; as a nutrient source for the growth ofother microbes or organisms; as feed for animals including but notlimited to cattle, sheep, chickens, pigs, or fish; as feed stock formethane or biogas production; as fertilizer; soil additives and soilstabilizers.

An additional feature of certain embodiments of the present inventionrelates to the optimization of oxyhydrogen microorganisms for carbondioxide capture, carbon fixation into organic compounds, and theproduction of other valuable chemical co-products. This optimization canoccur through methods known in the art of artificial breeding includingbut not limited to accelerated mutagenesis (e.g. using ultraviolet lightor chemical treatments), genetic engineering or modification,hybridization, synthetic biology or traditional selective breeding. Forcertain embodiments of the present invention utilizing a consortium ofmicroorganisms, the community can be enriched with desirable oxyhydrogenmicroorganisms using methods known in the art of microbiology throughgrowth in the presence of targeted electron donors including but notlimited to hydrogen, acceptors including but not limited to oxygen, andenvironmental conditions.

An additional feature of certain embodiments of the present inventionrelates to modifying biochemical pathways in oxyhydrogen microorganismsfor the production of targeted organic compounds. This modification canbe accomplished by manipulating the growth environment and/or throughmethods known in the art of artificial breeding including but notlimited to accelerated mutagenesis (e.g. using ultraviolet light orchemical treatments), genetic engineering or modification,hybridization, synthetic biology or traditional selective breeding. Theorganic compounds produced through the modification include but are notlimited to one or more of the following: biofuels including but notlimited to JP-8 jet fuel, diesel, gasoline, biodiesel, butanol, ethanol,long chain hydrocarbons, lipids, fatty acids, pseudovegetable oil, andmethane produced from biological reactions in vivo; or organic compoundsand/or biomass optimized as a feedstock for biofuel and/or liquid fuelproduction through chemical post-processing. These forms of fuel can beused as renewable/alternate sources of energy with low greenhouse gasemissions.

In order to give specific examples of the overall biological andchemical process for using oxyhydrogen microorganisms to capture CO₂ andproduce biomass and other useful co-products, a process flow diagramdescribing a specific embodiment of the present invention is nowprovided and described. This specific example should not be construed aslimiting the present invention in any way and is provided for the solepurpose of illustration.

FIG. 2 includes an exemplary process flow diagram illustrating oneembodiment of the present invention for the capture of CO₂ byoxyhydrogen microorganisms and the production of lipid rich biomass,which is converted to JP-8 jet fuel. In this set of embodiments, acarbon dioxide-rich flue gas is captured from an emission source such asa power plant, refinery, or cement producer. The flue gas can then becompressed and pumped into cylindrical anaerobic digesters containingone or more oxyhydrogen microorganisms such as but not limited to:purple non-sulfur photosynthetic bacteria including but not limited toRhodopseudomonas palustris, Rhodopseudomonas capsulata, Rhodopseudomonasviridis, Rhodopseudomonas sulfoviridis, Rhodopseudomonas blastica, andother Rhodopseudomonas sp.

In some embodiments, Rhodopseudomonas capsulata can be used as theoxyhydrogen microorganism, and, in some cases, a doubling time of 6hours for chemoautotrophic growth on hydrogen can be achieved. See, forexample, Madigan, Gest, J. Bacteriology (1979) 524-530, which isincorporated herein by reference. In some embodiments, the microbialdoubling time can be less than 6 hours, or shorter. In some embodiments,the dry biomass concentration can be at least about 3 g/l, at leastabout 4 g/l, or at least 5 g/1 at steady state. In some embodiments, thebiomass lipid content in the oxyhydrogen microorganism can be at leastabout 10%, at least about 20%, at least about 30%, at least about 35%,or at least about 40%. For example, in some embodiments,Rhodopseudomonas palustris can be used as the oxyhydrogen microorganism.See, for example, Carlozzi, Pintucci, Piccardi, Buccioni, Minieri,Lambardi, Biotechnol. Lett., (2009) DOI 10.1007/s10529-009-0183-2, whichis incorporated herein by reference. In certain embodiments, the biomasslipid content of the oxyhydrogen microorganisms is at least 40%; thereis a steady state bioreactor cell density of at least 5 g/liter in acontinuous process; the microbial doubling time is at most 6 hours; theprocess achieves at least a 40% energy efficiency in converting hydrogeninto biomass; and/or at least 60% of the biomass energy content isstored as lipid (which corresponds to about 40% biomass lipid content byweight).

In the set of embodiments illustrated in FIG. 2, hydrogen electron donorand oxygen and carbon dioxide electron acceptors are added continuouslyto the growth broth along with other nutrients required forchemosynthesis and culture maintenance and growth that are pumped intothe digester. In certain embodiments, the hydrogen source is a carbondioxide emission-free process. Exemplary carbon dioxide emission-freeprocesses include, for example, electrolytic or thermochemical processespowered by energy technologies including but not limited tophotovoltaics, solar thermal, wind power, hydroelectric, nuclear,geothermal, enhanced geothermal, ocean thermal, ocean wave power, tidalpower. In the set of embodiments illustrated in FIG. 2, oxygen serves asan electron acceptor in the chemosynthetic reaction for theintracellular production of ATP through the oxyhydrogen reaction linkedto oxidative phosphorylation. The oxygen can originate from the fluegas, it can be generated from the water-splitting reaction used toproduce the hydrogen, and/or it can be taken from air. In FIG. 2, carbondioxide from the flue gas serves as an electron acceptor for thesynthesis of organic compounds through biochemical pathways utilizingthe ATP produced through the oxyhydrogen reaction and NADH and/or NADPHproduced from the intracellular enzymatically catalyzed reduction ofNAD⁺ or NADP⁺ by H₂. The culture broth can be continuously removed fromthe digesters and flowed through membrane filters to separate the cellmass from the broth. The cell mass can then be recycled back into thedigesters and/or pumped to post-processing where lipid extraction isperformed according to methods known to those skilled in the art. Thelipids can then be converted to JP-8 jet fuel using methods known tothose skilled in the art of biomass refining (see, for example, U.S. DOEEnergy Efficiency & Renewable Energy Biomass Program, “National AlgalBiofuels Technology Roadmap”, May 2010, which is incorporated herein byreference in its entirety. Cell-free broth which has passed through thecell mass removing filters can then be subjected to any necessaryadditional waste removal treatments which depends on the source of fluegas. The remaining water and nutrients can then be pumped back into thedigesters.

Some of the Rhodopseudomonas species have extremely versatilemetabolisms, making them capable of photoautotrophic,photoheterotrophic, heterotrophic, as well as chemoautotrophic growthand the ability to live in both aerobic and anaerobic environments[Madigan, Gest, J. Bacteriology (1979) 524-530]. In certain embodimentsof the present invention the heterotrophic capability ofRhodopseudomonas sp. is exploited to further improve the efficiency ofenergy and carbon conversion to lipid product. The non-lipid biomassremainder following lipid extraction is composed of primarily proteinand carbohydrate. In certain embodiments of the present invention, someof the carbohydrate and/or protein remainder following lipid extractionis acid hydrolyzed to simple sugars and/or amino acids, the acid isneutralized, and the solution of simple sugars and/or amino acids arefed to a second heterotrophic bioreactor containing Rhodopseudomonas sp.that consumes the biomass input and produces additional lipid product,as illustrated in FIG. 2.

The Rhodopseudomonas palustris genome has been sequenced by the DOEJoint Genome Institute [Larimer et. al (2003) Nature Biotechnology 22,55-61]. It is reported that its genetic system is particularly amenableto modification. In one set of embodiments of the present invention thecarbon-fixation reaction or reactions are performed by Rhodopseudomonassp. that have been improved, optimized or engineered for the improvedfixation of carbon dioxide and/or other forms of inorganic carbon and/orthe improved production of organic compounds through methods includingbut not limited to one or more of the following: acceleratedmutagenesis, genetic engineering or modification, hybridization,synthetic biology or traditional selective breeding.

FIG. 3 includes an exemplary schematic diagram of a bioreactor 300,which can be used in certain embodiments. Bioreactor 300 can be used,for example, as the reactor illustrated as Box 4 in FIG. 1 labeled“Bioreactor—Knallgas Microbes” and/or as the reactor illustrated as Box4 in FIG. 2 labeled “Bioreactor—Purple non-sulfur bacterial.” Bioreactor300 illustrated in FIG. 3 can be operated to take advantage of the lowsolubilities of hydrogen and oxygen gas in water and avoids dangerousmixtures of hydrogen and oxygen gas. In addition, the bioreactor canprovide the oxyhydrogen microorganisms with the oxygen and hydrogenneeded for cellular energy and carbon fixation, for example, bysparging, bubbling, or diffusing oxygen or air up a vertical liquidcolumn filled with culture medium.

Bioreactor 300 includes a first column 302 and a second column 304. Inthe set of embodiments illustrated in FIG. 3, oxygen is introduced tofirst column 302 while hydrogen or syngas is introduced to second column304, although in other embodiments, their order may be reversed. Theoxygen and/or hydrogen and/or syngas can be introduced to theirrespective columns by, for example, sparging, bubbling, and/or diffusionsuch that they travel upwards through the culture medium. Bioreactor 300can include a horizontal liquid connection 312 at the top of the columnsand a horizontal liquid connection 314 at the bottom of the columns.

In some embodiments, the level of the liquid medium with column 302 ismaintained such that gaseous headspace 316 is formed above the liquid.In addition, in some cases, the level of liquid medium within column 304can be arranged such that gaseous headspace 318 is formed above theliquid medium. In some embodiments, headspace 316 and/or headspace 318can occupy at least about 2%, at least about 10%, at least about 25%,between 2% and about 80%, between about 10% and about 80%, or betweenabout 25% and about 80% of the total volume of the column in which theyare positioned. Headspaces 316 and 318 can be isolated from each otherby the liquid medium. In some embodiments, the low solubility of thegases in the liquid medium allow for the collection of gases at the topsof the columns after bubbling or diffusing the gases up through theirrespective columns. Establishing isolated headspaces can prevent adangerous amount of hydrogen and oxygen gases from mixing with eachother. For example, the hydrogen gas in one column can be prevented frommixing with the oxygen gas in other column (and vice versa) Inhibitingmixing of the hydrogen and oxygen gases can be achieved, for example, bymaintaining the connections between the two columns such that they arefilled with liquid, thereby preventing transport of the gases from onecolumn to the other. In some embodiments, headspaces 316 and/or 318 canremain substantially stationary at the top of their respective columnsas liquid medium is circulated between the first and second columns.

In FIG. 3, the horizontal liquid connection 312 at the top of thecolumns and horizontal liquid connection 314 bottom of the columns arearranged such that they allow the liquid medium to flow up one column inthe direction of the oxygen gas, and down the other column,countercurrent to the hydrogen gas and/or syngas while the horizontalliquid connections remain continuously filled. In other embodiments, theliquid medium can flow up the column containing the hydrogen gas and/orsyngas and down the other column containing the oxygen gas(incountercurrent flow relative to the gas).

In some embodiments, the gas on one side or the other, but not bothsides simultaneously, may be bubbled forcefully such that thatparticular column acts as an airlift reactor and drives the circulationof the culture medium between the two columns. In some embodiments, thecirculation of the fluid may also be assisted by impellers, turbines,and/or pumps.

In some such embodiments, any unused hydrogen gas and/or syngas thatpasses through the culture medium without being taken up by themicroorganisms (and which may end up in the head space) can berecirculated by pumping the gas out of the headspace, optionallycompressing it, and pumping it back into the medium at the bottom of theliquid column on the hydrogen and/or syngas side. In some embodiments,the oxygen and/or air might be similarly be recirculated on itsrespective side or alternatively vented after passing through theheadspace.

The oxyhydrogen microorganisms are allowed to freely circulate alongwith the liquid medium between the first and second columns in certainembodiments. In other embodiments, the oxyhydrogen microorganisms arerestricted to the hydrogen side, for example, by using a microfilterthat retains the microorganisms on the hydrogen side but allows theliquid medium to pass through.

FIG. 4 includes an exemplary schematic illustration of another method ofoperating bioreactor 300 that can be used in certain embodiments. Thebioreactor arrangement in FIG. 4 can take advantage of the relativelyhigh solubility of carbon dioxide and/or the strong ability ofoxyhydrogen microorganism to capture carbon dioxide from relativelydilute streams. The operation illustrated in FIG. 4 can exploit thecarbon concentrating mechanism native to oxyhydrogen microorganisms.Flue gas and/or air containing carbon dioxide can be transported throughthe oxygen side of the bioreactor. The carbon dioxide can be dissolvedinto solution and/or taken up by the oxyhydrogen microbes andsubsequently transported over to the hydrogen side of the reactor, forexample, through the horizontal liquid connection 312 at the top of thecolumn On the hydrogen side, reducing equivalents can be provided thatdrive fixation of the carbon. In some embodiments, other gases pumped inon the oxygen side (e.g., oxygen, nitrogen, etc.) have a low solubilityrelative to CO₂, and are not carried over to the hydrogen side. Ratherthan being passed from column 302 to column 304, the low solubilitygases can be transported to headspace 316. In some embodiments, afterthe gases are transported to headspace 316, they can be vented.

FIG. 5 includes an exemplary schematic diagram of an electrolysisapparatus 500, which can be used in certain embodiments. Electrolysisapparatus 500 can be used, for example, as the unit illustrated as Box 3in FIG. 1 labeled “Electron donor generation” and/or as the unitillustrated as Box 3 in FIG. 2 labeled “Electrolysis.” Electrolysisapparatus 500 can be designed to take advantage of the oxyhydrogenmicroorganisms' tolerance and need for a certain concentration of oxygenby decreasing or eliminating the complete separation of the hydrogen andoxygen produced from the electrolysis step, relative to the separationschemes employed in conventional electrolysis systems designed for theproduction of pure hydrogen. Apparatus 500 includes an electrolysis unit502 that is configured to generate H₂ and O₂ from water. Any suitableelectrolysis unit 502 can be employed to perform the electrolysis step.In some embodiments, the electrical resistance in electrolysis unit 502can be reduced at the expense of complete hydrogen and oxygen separationby means including but not limited one or more of the following:removing the separator used to prevent gas crossover in standardelectrolyzers and/or using a relatively short distance between positiveand negative electrodes.

Apparatus 500 can include an outlet 504, through which the hydrogen andoxygen produced by the electrolysis unit 502 can be transported. Outlet504 can be equipped with a separator 506, which can be used to separateat least a portion of the hydrogen from at least a portion of theoxygen. In certain embodiments, semipermeable membranes such as polymermembranes designed for H₂ separation can be employed as separator 506.In certain embodiments, separator 506 can include metal foils includingbut not limited to foils made from palladium, palladium alloys,vanadium, niobium, tantalum and their alloys, and/or other metals and/oralloys that are permeable to hydrogen but less permeable to other gasessuch as oxygen. In some embodiments, the separator can be used toseparate the hydrogen from the oxygen such that the hydrogen content ofone gas product exiting the separator is enriched to a level that isdesirable for oxyhydrogen microbes. The gas product can then betransported to a bioreactor, where it can be used as a feedstock. Incertain embodiments, the amount of hydrogen in one of the gas productsexiting the separator can be set at a level such that oxyhydrogenmicroorganism activity is maximized, and the loss of hydrogen producedthrough electrolysis apparatus 500 is minimized

The following documents are incorporated herein by reference in theirentirety for all purposes: U.S. Provisional Patent Application No.61/328,184, filed Apr. 27, 2010 and entitled “USE OF OXYHYDROGENMICROORGANISMS FOR NON-PHOTOSYNTHETIC CARBON CAPTURE AND CONVERSION OFINORGANIC CARBON SOURCES INTO USEFUL ORGANIC COMPOUNDS”; InternationalPatent Application Serial No. PCT/US2010/001402, filed May 12, 2010,entitled “BIOLOGICAL AND CHEMICAL PROCESS UTILIZING CHEMOAUTOTROPHICMICROORGNISMS FOR THE CHEMOSYTHETIC FIXATION OF CARBON DIOXIDE AND/OROTHER INORGANIC CARBON SOURCES INTO ORGANIC COMPOUNDS, AND THEGENERATION OF ADDITIONAL USEFUL PRODUCTS”; and U.S. Patent ApplicationPublication No. 2010/0120104, filed Nov. 6, 2009, entitled “BIOLOGICALAND CHEMICAL PROCESS UTILIZING CHEMOAUTOTROPHIC MICROORGNISMS FOR THECHEMOSYTHETIC FIXATION OF CARBON DIOXIDE AND/OR OTHER INORGANIC CARBONSOURCES INTO ORGANIC COMPOUNDS, AND THE GENERATION OF ADDITIONAL USEFULPRODUCTS.

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

Example 1

In this example, oxyhydrogen microorganisms that accumulate high lipidcontent and/or other valuable compounds such as polyhydroxybutyrate(PHB) to are grown on an inorganic medium with CO₂ as the carbon sourceand hydrogen acting as the electron donor while oxygen provides theelectron acceptor. Oxyhydrogen microbes such as these can be used incertain embodiments of the present invention in converting C1 chemicalssuch as carbon dioxide into longer chain organic chemicals.

Static anaerobic reaction vessels were inoculated with Cupriavidusnecator DSM 531 (which can accumulate a high percentage of cell mass asPHB). The inoculum were taken from DSM medium no. 1 agar plates keptunder aerobic conditions at 28 degrees Celsius. Each anaerobic reactionvessel had 10 ml of liquid medium DSM no. 81 with 80% H₂, 10% CO₂ and10% O₂ in the headspace. The cultures were incubated at 28 degreesCelsius. The Cupriavidus necator reached an optical density (OD) at 600nm of 0.98 and a cell density of 4.7×10⁸ cells/ml after 8 days.

Another growth experiment was performed for Cupriavidus necator (DSM531). The medium used for growth was the mineral salts medium (MSM)formulated by Schlegel et al. The MSM medium was formed by mixing 1000ml of Medium A, 10 ml of Medium B, and 10 ml of Medium C. Medium Aincluded 9 g/l Na₂HPO₄.12H₂O, 1.5 g/l KH₂PO₄, 1.0 g/l, 0.2 g/lMgSO₄.7H₂O, and 1.0 ml of Trace Mineral Medium. The Trace Mineral Mediumincluded 1000 ml distilled water; 100 mg/l ZnSO₄.7H₂O; 30 mg/l MnCl₂.4H₂O; 300 mg/l H₃BO₃; 200 mg/l COCl₂.6H₂O; 10 mg/l CuCl₂.2 H₂O; 20 mg/lNiCl₂.6H₂O; and 30 mg/l Na₂MoO₄.2H₂O. Medium B contained 100 ml ofdistilled water; 50 mg ferric ammonium citrate; and 100 mg CaCl₂. MediumC contained 100 ml of distilled water and 5 g NaHCO₃.

The cultures were grown in 20 ml of MSM media in 150-ml stopped andsealed serum vials with the following gas mixture in the headspace: 71%Hydrogen; 4% Oxygen; 16% Nitrogen; 9% Carbon dioxide. The headspacepressure was 7 psi. The cultures were grown for eight days at 30 degreesCelsius. Cupriavidus necator reached an OD at 600 nm of 0.86.

It is known that, in larger scale bioreactor equipment, faster growthrates and higher cell densities can be attained. Accordingly, it isbelieved that higher growth rates and cell densities can be achievedsimply by scaling up the systems described above. For exampleCupriavidus necator which is also known as Alcaligenes eutrophus,Ralstonia eutropha, Hydrogenomona eutropha, has been grown inbioreactors on H₂/CO₂/O₂ to a cell density of over 90 grams/liter[Tanaka, Ishizaki; Biotech. And Bioeng., vol. 45, 268-275 (1995)], andwith doubling times below two hours [Ammann, Reed, Durichek, Appl.Microbio., (1968) 822-826].

Specific preferred embodiments of the present invention have beendescribed here in sufficient detail to enable those skilled in the artto practice the full scope of invention. However it is to be understoodthat many possible variations of the present invention, which have notbeen specifically described, still fall within the scope of the presentinvention and the appended claims. Hence these descriptions given hereinare added only by way of example and are not intended to limit, in anyway, the scope of this invention. More generally, those skilled in theart will readily appreciate that all parameters, dimensions, materials,and configurations described herein are meant to be exemplary and thatthe actual parameters, dimensions, materials, and/or configurations willdepend upon the specific application or applications for which theteachings of the present invention is/are used. Those skilled in the artwill recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. It is, therefore, to be understood that theforegoing embodiments are presented by way of example only and that,within the scope of the appended claims and equivalents thereto, theinvention may be practiced otherwise than as specifically described andclaimed. The present invention is directed to each individual feature,system, article, material, kit, and/or method described herein. Inaddition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively.

What is claimed is:
 1. A biological and chemical method for the capture and conversion of an inorganic carbon compound and/or an organic compound containing only one carbon atom into an organic chemical product, comprising: introducing an inorganic carbon compound and/or an organic compound containing only one carbon atom into an environment suitable for maintaining oxyhydrogen microorganisms and/or capable of maintaining extracts of oxyhydrogen microorganisms; and converting the inorganic carbon compound and/or the organic compound containing only one carbon atom into the organic chemical product and/or a precursor thereof within the environment via at least one chemosynthetic carbon-fixing reaction utilizing the oxyhydrogen microorganisms and/or cell extracts containing enzymes from the oxyhydrogen microorganisms; wherein the chemosynthetic fixing reaction is at least partially driven by chemical and/or electrochemical energy provided by electron donors and electron acceptors that have been generated chemically and/or electrochemically and/or are introduced into the environment from at least one source external to the environment.
 2. A method according to claim 1, wherein the inorganic carbon compound comprises carbon dioxide.
 3. A method according to claim 1, wherein the carbon dioxide comprises carbon dioxide gas, either alone and/or dissolved in a mixture or solution further comprising carbonate ion and/or bicarbonate ion.
 4. A method according to claim 1, wherein the inorganic carbon comprises inorganic carbon contained in a solid phase.
 5. A method according to claim 1, wherein the organic compound containing only one carbon atom comprises carbon monoxide, methane, methanol, formate, and/or formic acid.
 6. A method according to claim 1, wherein said electron donors and/or organic compounds containing only one carbon atom are generated through the gasification and/or pyrolysis of organic matter and provided as a syngas to the oxyhydrogen microorganisms.
 7. A method according to claim 1, wherein said electron donors and/or organic compounds containing only one carbon atom are generated through methane steam reforming and provided as a syngas to the oxyhydrogen microorganisms.
 8. A method according to claim 6, wherein the ratio of hydrogen to carbon monoxide in the syngas is adjusted via the water gas shift reaction prior to the syngas being delivered to the oxyhydrogen microorganisms.
 9. A method according to claim 1, wherein the oxyhydrogen microorganisms include oxyhydrogen microorganisms selected from one or more of the following categories: purple non-sulfur photosynthetic bacteria, cyanobacteria, and/or green algae.
 10. A method according to claim 1, wherein the oxyhydrogen microorganisms include oxyhydrogen microorganisms selected from cyanobacteria and/or green algae.
 11. A method according to claim 1, wherein the oxyhydrogen microorganisms include oxyhydrogen microorganisms selected from one or more of the following genera: Rhodopseudomonas sp.; Rhodospirillum sp.; Rhodococcus sp.; Rhizobium sp.; Thiocapsa sp.; Pseudomonas sp.; Hydrogenomonas sp.; Hydrogenobacter sp.; Hydrogenovibrio sp.; Helicobacter sp.; Xanthobacter sp.; Hydrogenophaga sp.; Bradyrhizobium sp.; Ralstonia sp.; Alcaligenes sp.; Variovorax sp.; Acidovorax sp.; Anabaena sp.; Scenedesmus sp.; Chlamydomonas sp., Ankistrodesmus sp., and Rhaphidium sp.
 12. A method according to claims 1, wherein the oxyhydrogen microorganisms include oxyhydrogen microorganisms selected from one or more of the following genera: Rhodospirillum sp.; Rhizobium sp.; Thiocapsa sp.; Hydrogenovibrio sp.; Helicobacter sp.; Xanthobacter sp.; Hydrogenophaga sp.; Bradyrhizobium sp.; Variovorax sp.; Acidovorax sp.; Anabaena sp.; Scenedesmus sp.; Chlamydomonas sp., Ankistrodesmus sp., and Rhaphidium sp.
 13. A method according to claim 1, wherein said electron donors include but are not limited to one or more of the following reducing agents: ammonia; ammonium; carbon monoxide; dithionite; elemental sulfur; hydrocarbons; hydrogen; metabisulfites; nitric oxide; nitrites; sulfates such as thiosulfates including but not limited to sodium thiosulfate (Na₂S₂O₃) or calcium thiosulfate (CaS₂O₃); sulfides such as hydrogen sulfide; sulfites; thionate; thionite; transition metals or their sulfides, oxides, chalcogenides, halides, hydroxides, oxyhydroxides, phosphates, sulfates, or carbonates, in dissolved or solid phases; and conduction or valence band electrons in solid state electrode materials.
 14. A method according to claim 1, wherein said electron acceptors comprise one or more of the following: carbon dioxide; oxygen; nitrites; nitrates; ferric iron or other transition metal ions; sulfates; or valence or conduction band holes in solid state electrode materials.
 15. A method according to claim 1, wherein the converting step is preceded by one or more chemical preprocessing steps in which said electron donors and/or said electron acceptors are generated and/or refined from at least one input chemical and/or are recycled from chemicals produced during the fixing step and/or chemicals derived from waste streams from other industrial, mining, agricultural, sewage or waste generating processes.
 16. A method according to claim 1, wherein the converting step is followed by one or more process steps in which organic and/or inorganic chemical products of chemosynthesis are separated from a process stream produced during the converting step and processed to form products in a form suitable for storage, shipping, and sale; as well as one or more process steps in which cell mass is separated from the process stream and recycled to the environment as and/or collected and processed to produce biomass in a form suitable for storage, shipping, and sale. 17-25. (canceled)
 26. A bioreactor, comprising: a first column comprising an upper portion and a lower portion; a second column comprising an upper portion and a lower portion, the upper portion of the second column fluidically connected to the upper portion of the first column, and the lower portion of the second column fluidically connected to the lower portion of the first column; wherein, the bioreactor is constructed and arranged such that, when a liquid is circulated between the first and second columns, a volume of gas is substantially stationary at the top of the first column and/or the second column, and the volume of gas occupies at least about 2% of the total volume of the column in which the volume is positioned. 27-28. (canceled)
 29. A method of operating a bioreactor, comprising circulating a liquid comprising a growth medium between a first column and a second column, wherein, during operation, a volume of gas remains substantially stationary at the top of the first column and/or the second column, and the volume of gas occupies at least about 2% of the total volume of the column in which the volume is positioned. 30-34. (canceled)
 35. An electrolysis device, comprising: a chamber constructed and arranged to electrolyze water to produce oxygen and hydrogen; and an outlet comprising a separator constructed and arranged to separate at least a portion of the oxygen within a stream from at least a portion of the hydrogen within a stream such that the hydrogen content of the fluid exiting the separator is suitable for use as a feed stream to a reactor containing a culture of oxyhydrogen microorganisms. 36-37. (canceled)
 38. A method of operating an electrolysis device, comprising: electrolyzing water to produce a first stream containing oxygen and hydrogen; and separating at least a portion of the oxygen from at least a portion of the hydrogen to produce a second stream relatively rich in hydrogen compared to the first stream, wherein the second stream is suitable for use as a feed stream to a reactor containing a culture of oxyhydrogen microorganisms. 39-40. (canceled) 